EP2275117A1 - GLP-1, exendine-4, analogues de peptides et utilisations associées - Google Patents

GLP-1, exendine-4, analogues de peptides et utilisations associées Download PDF

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EP2275117A1
EP2275117A1 EP10177860A EP10177860A EP2275117A1 EP 2275117 A1 EP2275117 A1 EP 2275117A1 EP 10177860 A EP10177860 A EP 10177860A EP 10177860 A EP10177860 A EP 10177860A EP 2275117 A1 EP2275117 A1 EP 2275117A1
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glp
seq
exendin
cells
polypeptide
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EP2275117B1 (fr
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Nigel H. Greig
Josephine Egan
Maire Doyle
Harold Holloway
Tracy Ann Perry
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US Department of Health and Human Services
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    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A61K38/22—Hormones
    • A61K38/2278—Vasoactive intestinal peptide [VIP]; Related peptides (e.g. Exendin)
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons

Definitions

  • This invention relates generally to glucagon-like peptide-1 (GLP-1), exendin-4 and their peptide analogs.
  • GLP-1 glucagon-like peptide-1
  • exendin-4 exendin-4
  • peptide analogs a group consisting of glucagon-like peptide-1 (GLP-1), exendin-4 and their peptide analogs.
  • the invention also relates to their uses in the treatment of diabetes and neurodegenerative conditions.
  • pancreatic beta cell dysfunction and the concomitant decrease in insulin production can result in diabetes mellitus.
  • type 1 diabetes the beta cells are completely destroyed by the immune system, resulting in an absence of insulin producing cells (Physician's Guide to Insulin Dependent [Type I] Diabetes Mellitus: Diagnosis and Treatment, American Diabetes Association, 1988).
  • type 2 diabetes the beta cells become progressively less efficient as the target tissues become resistant to the effects of insulin on glucose uptake. Thus, beta cells are absent in people with type 1 diabetes and are functionally impaired in people with type 2 diabetes.
  • Beta cell dysfunction currently is treated in several different ways.
  • insulin replacement therapy is necessary. Insulin therapy, although life-saving, does not restore normoglycemia, even when continuous infusions or multiple injections are used in complex regimes. For example, postprandial levels of glucose continue to be excessively high in individuals on insulin replacement therapy. Thus, insulin therapy must be delivered by multiple daily injections or continuous infusion and the effects must be carefully monitored to avoid hyperglycemia, hypoglycemia, metabolic acidosis, and ketosis.
  • GLP-1 Glucagon-like peptide-1
  • type 2 diabetes Gutniak et al., 1992; Nauck et al., J. Clin. Invest., 1993 ). It increases insulin release by the beta cells even in subjects with long-standing type 2 diabetes ( Nauck et al., Diabetologia, 1993 ).
  • GLP-1 treatment has an advantage over insulin therapy because GLP-1 stimulates endogenous insulin secretion, which turns off when blood glucose levels drop ( Nauck et al., Diabetologia, 1993 ; Elahi et al., 1994).
  • GLP-1 promotes euglycemia by increasing insulin release and synthesis, inhibiting glucagon release, and decreasing gastric emptying ( Nauck et al., Diabetologia, 1993 ; Elahi et al., 1994; Wills et al., 1996; Nathan et al., 1992; De Ore et al., 1997). GLP-1 also induces an increase in hexokinase messenger RNA levels ( Wang et al., Endocrinology 1995 ; Wang et al., 1996).
  • GLP-1 is known to have a potent insulin-secreting effect on beta cells (Thorens and Waeber, 1993; Orskov, 1992) and to increase insulin biosynthesis and proinsulin gene expression when added to insulin-secreting cell lines for 24 hours (Drucker et al., 1987; Fehmann and Habener, 1992). In studies using RIN 1046-38 cells, twenty-four hour treatment with GLP-1 increased glucose responsiveness even after the GLP-1 had been removed for an hour and after several washings of the cells (Montrose-Rafizadeh et al., 1994). Thus, GLP-1 is an insulinotropic agent known to have biological effects on ⁇ cells even after it has been metabolized from the system.
  • GLP-1 is a product of posttranslational modification of proglucagon.
  • the sequences of GLP-1 and its active fragments GLP-1 (7-37) and GLP-1(7-36) amide are known in the art (Fehmann et al., 1995).
  • GLP-1 has been proposed as a therapeutic agent in the treatment of diabetes, it has a short biological half-life (De Ore et al., 1997), even when given by a bolus subcutaneously (Ritzel et al., 1995).
  • GLP-1 degradation (and GLP-1 (7-36) amide), in part, is due to the enzyme dipeptidyl peptidase (DPP1V), which cleaves the polypeptide between amino acids 8 and 9 (alanine and glutamic acid).
  • DPP1V dipeptidyl peptidase
  • Exendin-4 is a polypeptide produced in the salivary glands of the Gila Monster lizard (Goke et al., 1993).
  • the amino acid sequence for exendin-4 is known in the art (Fehmann et al. 1995). Although it is the product of a uniquely non-mammalian gene and appears to be expressed only in the salivary gland (Chen and Drucker, 1997), exendin-4 shares a 52% amino acid sequence homology with GLP-1 and in mammals interacts with the GLP-1 receptor (Goke et al., 1993; Thorens et al., 1993).
  • exendin-4 has been shown to promote insulin secretion by insulin producing cells and, given in equimolar quantities, is more potent than GLP-1 at causing insulin release from insulin producing cells. Furthermore, exendin-4 potently stimulates insulin release to reduce plasma glucose levels in both rodents and humans and is longer acting than GLP-1. Exendin-4, however, because it does not occur naturally in mammalians, has certain potential antigenic properties in mammals that GLP-1 lacks.
  • peripheral neuropathy In addition to the reduction in insulin production that occurs in diabetes, peripheral neuropathy is commonly associated with diabetes. Twenty to thirty percent of all diabetes subjects eventually develop peripheral neuropathy. Furthermore, there are reports of increased risk of Alzheimer's disease with heart disease, stroke, hypertension, and diabetes (Moceri et al., 2000; Ott et al., 1999). Thus, diabetes is a disease that is also associated with neurodegenerative diseases.
  • GLP-1 receptor is present in both the rodent (Jin et al 1988, Shughrue et al 1996) and human (Wei and Mojsov 1995, Satoh et al 2000) brains.
  • the chemoarchitecture of the distribution appears to be largely confined to the hypothalamus, thalamus, brainstem, lateral septum, the subfornical organ and the area postrema, all circumventricular areas where generally large numbers of peptide receptors are located.
  • specific binding sites for GLP-1 have also been detected throughout the caudate-putamen, cerebral cortex and cerebellum (Campos et al. 1994, Calvo et al. 1995, Goke et al. 1995), albeit at low densities.
  • polypeptides that are of therapeutic value in the treatment of diabetes and the treatment of degenerative disorders such as Alzheimer's and Parkinson's diseases, as well as the peripheral neuropathy associated with type 2 diabetes mellitus.
  • this invention in one aspect, relates to novel polypeptide analogues of GLP-1 and exendin-4.
  • the polypeptide in a preferred embodiment, is insulinotropic and long-acting.
  • the polypeptide's insulinotropic effect is comparable to or exceeds the effect of an equimolar amount of GLP-1 or exendin-4.
  • the invention further relates to a purified polypeptide, the amino acid sequence of which comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:25, or SEQ ID NO:33.
  • the invention in another aspect, relates to a method of treating a subject with diabetes, comprising administering to the subject the polypeptide of the invention in an amount that has an insulinotropic effect.
  • Figure 1 shows sequences for the 35 synthetic polypeptides tested for their insulinotropic properties and the sequences for GLP-1 and Ex-4. Dark shading shows exendin-4-like residues and light shading shows GLP-1-like residues.
  • Figure 2 shows a comparison in insulin secretion in RIN 1048-36 cells in the presence of GLP-1, exendin-4, and the synthetic polypeptides identified in Figure 1 . Levels are expressed as a percentage of basal levels.
  • Figure 3 shows a comparison in insulin secretion in RIN 1048-38 cells in the presence of glucose (5mM) and in the presence or absence of 10 nM GLP-1 (SEQ ID NO:1, GLP-1 Gly 8 (peptide 1; SEQ ID NO:3), GLP-1 6-aminohexanoic acid 8 (peptide 11; SEQ ID NO:8), GLP-1 (6-aminohexanoic acid 9 ) 4 (peptide 25; SEQ ID NO:22),GLP-1 (6-aminohexanoic acid 9 ) 8 (peptide 26; SEQ ID NO: 23), or six analogues of GLP-1 that contain, from the carboxy terminus, 3, 5, 7, 12, 21, and all D-amino acids.
  • the data represent the mean of 2-3 experiments ⁇ SEM. **p ⁇ 0.001, *p ⁇ 0.05 for treated versus basal. Levels are expressed in pg of insulin/ ⁇ g of protein. Basal release is also shown.
  • Figure 4 shows the effect of GLP-1 analogs on the production of intracellular cAMP.
  • CHO/GLP-1R cells were incubated with the indicated polypeptides (10nM) for 30 min at 37°C, after which they were lysed and the lysates processed for determination of cAMP content.
  • the data are normalized to maximum values obtained in the presence of GLP-1 (10nM).
  • the data points represent the mean of 2-3 experiments. **p ⁇ 0.001, *p ⁇ 0.05 for treated versus basal.
  • Figure 5 shows dose response curves for GLP-1, GLP-1 Gly 8 (SEQ ID NO:3), and GLP-1 6-aminohexanoic acid 8 (SEQ ID NO:8).
  • Intracellular cAMP levels were measured in CHO/GLP-1R cells after treatment with the indicated concentrations of GLP-1, GLP-1 Gly 8 , and GLP-1 6-aminohexanoic acid 8 for 30 min at 37°C. The data were normalized to maximum values obtained in each experiment in the presence of
  • GLP-1 (10nM). Bars represent the means ⁇ SEM of three experiments preformed in triplicate.
  • Figure 6 shows the displacement of [ 125 I]-GLP-1 binding to CHO/GLP-1R cells with analogs of GLP-1.
  • [ 125 I]-GLP-1 binding to intact CHO/GLPR cells was competed with various concentrations of the polypeptides shown.
  • the data are normalized to maximum values obtained in the presence of 10nM of the respective polypeptides.
  • the data points represent the mean ⁇ SEM of three experiments preformed in triplicate.
  • Figure 7 shows the acute insulin-secreting activity of 0.4 nmol/kg of polypeptide Ex-4 WOT (SEQ ID NO:7) and GLP-1 Gly 8 (SEQ ID NO:3) in fasted, diabetic Zucker rats to induce insulin secretion as compared to equimolar concentrations of exendin-4 and GLP-1.
  • Figure 8 shows the time course of insulin-secreting activity of 0.4 nmol/kg of polypeptide 10 (Ex-4 WOT (SEQ ID NO:7)), polypeptidel (GLP-1 Gly 8 (SEQ ID NO:3)), and polypeptide11(GLP-1 6-aminohexanoic acid 8 (SEQ ID NO:8)) in fasted, diabetic Zucker rats up to 24 hours as compared to equimolar concentrations of exendin-4 and GLP-1.
  • polypeptide 10 Ex-4 WOT (SEQ ID NO:7)
  • polypeptidel GLP-1 Gly 8
  • SEQ ID NO:8 polypeptide11(GLP-1 6-aminohexanoic acid 8
  • Figure 9 shows the biological effects of GLP-1 Gly 8 (SEQ ID NO:3) and GLP-1 6-aminohexanoic acid 8 (SEQ ID NO:8).
  • Figure 9A shows the effect on blood glucose levels and
  • Figure 10 shows the displacement of [ 125 I] GLP-1binding to CHO/GLP-1R cells with the analogs of GLP-1, GLP-1 Gly8 and Ex-4.
  • [ 125 I] GLP-1 binding to intact CHO/GLP-1R cells was competed with various concentrations of the peptides shown.
  • Each of Figure 10A , B, and C show the data for different peptides. The data are normalized to maximum values obtained in the presence of 10nM of the respective peptides. The data points respresent the mean of three experiments performed in triplicate. B o , maximum binding in the absence of cold peptide.
  • Figure 11 shows the densitometric quantification of proteins extracted from NGF, exendin-4, exendin-4 WOT and GLP-1 treated PC12 cells.
  • Protein bands obtained from cell lysates and conditioned media samples were analyzed by Western blotting and immunoprobed with the 22C11 monoclonal antibody (epitope: ⁇ APP aa 66-81, Roche Molecular Biochemicals, Indianapolis, IN).
  • Data are presented as the percent change in expression of ⁇ APP derivatives from cell lysates samples (A and B) and soluble sAPP from conditioned media samples taken on day 3 of treatment (C and D) relative to untreated control samples cultured in low serum media alone.
  • Vertical error bars represent standard error of 3 individual experimental values.
  • Significant difference from untreated * p ⁇ 0.05 and ** p ⁇ 0.01.
  • Figure 12 shows the effect of different concentrations of NGF and/or exendin-4 treatment on neurite outgrowth in PC12 cells.
  • Neurite outgrowth is represented as the percent increase in number of cells bearing neurites relative to untreated (low serum medium).
  • Vertical error bars represent ⁇ standard error of the difference between the means of six individual experimental values.
  • Significant difference from untreated * p ⁇ 0.05 and ** p ⁇ 0.01.
  • Figure 13 shows the effect of exendin-4 treatment on NGF-mediated cell death. Combination treatments were carried out for a total of 7 days, in the presence or absence of 50 ng/ml NGF, with or without exendin-4 (at 1 or 5 mg/ml). Cells were subsequently harvested and allowed to rejuvenate in complete media for an additional 3 days. Cell survival is presented as the proportion of viable cells (by MTT method) on day 10. Vertical error bars represent ⁇ standard error of four individual experimental values.
  • Figure 14 shows densitometric quantification of the synaptophysin protein extracted from NGF, exendin-4, exendin-4 WOT and GLP-1 treated PC12 cells. Protein bands obtained from cell lysate samples were analyzed by Western blotting and immunoprobed with the synaptophysin monoclonal antibody, which stains neurosecretory vesicles. Synaptophysin was used as a marker of differentiation. Density of the synaptophysin protein is presented as the percent difference from untreated. Vertical error bars represent ⁇ standard error of three individual experimental values conducted at separate time intervals. Significant difference from untreated: ** p ⁇ 0.01.
  • Figure 15 shows fold increases in lactate dehydrogenase (LDH) levels in the conditioned medium of PC12 cells following treatment with NGF, exendin-4, exendin-4 WOT and GLP-1.
  • LDH levels are a marker of cell viability, with elevated levels being associated with a loss of cell integrity.
  • Vertical error bars represent ⁇ standard error of the difference between the means of three individual experimental values conducted at separate time intervals. Significant difference from untreated: * p ⁇ 0.05 and ** p ⁇ 0.01.
  • Figure 16 shows displacement of 125 I - GLP-1 binding with cold GLP-1 (A), GLP-1 stimulated release of cAMP (B) and protection against glutamate-induced apoptosis (C) in cultured hippocampal neurons.
  • 125 I-GLP-1 binding to intact cultured hippocampal neurons was competed with various concentrations of GLP-1.
  • the data are normalized to maximum values obtained in the presence of 1 ⁇ M GLP-1. Each data point represents the mean of two experimental values and is presented as the percentage of maximum binding in the absence of cold peptide.
  • cAMP levels were assayed over 30 min incubation with 10 nM GLP-1 (B). Vertical error bars represent ⁇ standard error of the mean of three individual experimental values.
  • Figure 17 shows choline acetyltransferase and glial fibrillary acidic protein immunoreactivity in the basal nucleus.
  • Panels A & B and C & D depict the left and right basal nucleus from individual animals which received vehicle infusion and GLP-1 infusion, respectively.
  • ChAT - positive immunoreactivity in the ipsilateral basal nucleus in an animal which received vehicle infusion (A) was substantially lower than that in the ipsilateral basal nucleus in an animal which received GLP-1 infusion (C).
  • Glial fibrillary acidic protein (GFAP) immunoreactivity a marker for reactive astrocytes produced in response to injury, demonstrated areas of positive immunoreactivity surrounding the site of cannula implantation and lining of the lateral ventricle in the vicinity of the site of infusion.
  • infusion of GLP-1 produced an elevated glial reaction in the basal nucleus on the infusion side (F) than that apparent as a result of the lesion (E) or after vehicle infusion.
  • Figure 18 shows percentage of difference in the Abercrombie corrected number of ChAT-immunoreactive cell bodies in the ipsilateral basal nucleus (lesion side) relative to the intact contralateral basal nucleus in sham and ibotenic acid animals receiving intracerebroventricularly (i.c.v.) infusion of vehicle (artifical CSF: aCSF), exendin-4 or GLP-1.
  • Vertical error bars represent the standard error of the difference between the means.
  • Significant difference from ibotenic acid vehicle group * p ⁇ 0.05 and ** p ⁇ 0.01.
  • Figure 19 shows that treatment of PC12 cells with GLP-1 and analogues significantly decreased ⁇ APP and sAPP protein levels without cellular dysfunction.
  • Treatment with NGF, exendin-4, exendin-4 WOT and GLP-1 was not associated with cellular dysfunction as determined by measurement of LDH levels from conditioned media samples compared with media standards (panel A).
  • Densitometric quantification of the immunoprobed proteins are presented as the mean percent change in expression of ⁇ APP derivatives from cell lysates samples (panel B) and soluble sAPP from conditioned media samples (panel C) taken on day 3 of treatment relative to untreated control samples cultured in low serum media alone.
  • the treatment conditions illustrated along the x-axis are common to panels A, B and C. Vertical error bars represent standard error of 3 individual experimental values. Significant difference from untreated: * p ⁇ 0.05 and ** p ⁇ 0.01.
  • Figure 20 shows GLP-1 treatment significantly reduced endogenous A ⁇ 1-40 levels in control mice.
  • Control mice were infused i.c.v. with GLP-1 (3.3 ⁇ g and 6.6 ⁇ g), exendin-4 (0.2 ⁇ g), NGF (2 ⁇ g) and control (vehicle).
  • Biochemical analysis of whole brain homogenates was carried out by sandwich ELISA for A ⁇ 1-40.
  • a ⁇ values are expressed as the mean A ⁇ concentration in fmol / g ⁇ SEM from treated and untreated animals. Significant difference from control: ** p ⁇ 0.01.
  • Figure 21 shows dose response curves for some of the Ex-4 and GLP-1 Gly8 analogs. Intracellular cAMP levels were measured in Rin 1046-38 cells after treatment with the indicated concentrations of the peptides for 30 min at 37 C. The data are normalized to maximum values obtained in each experiment for each peptide.
  • Figure 22 shows the acute biological effects of the peptides on blood glucose levels.
  • the results with Ex(1-36) (circle) and Ex(1-35)(square) are shown in Figure 22A , and the results with additional peptides are shown Figure 22B .
  • Figure 23 shows the abdominal fat volume lost over 51 days in control (white bars) and Ex (1-30) treated animals (black bars). The values are expressed as a percentage of the initial total fat volume (0 days). For each group, the total fat lost, as well as the fat lost from the visceral and subcutaneous tissue fractions is shown. The data shows that there was a significantly greater volume of total fat and visceral fat lost in the Ex(1-30) treated animals than in the control animals. Both groups showed decreased total fat volume at the 51 day time point. In the control group, this loss was largely due to loss of fat from the subcutaneous fraction, which occurred to a similar extent in the treated animals. *P ⁇ 0.05 Ex(1-30) vs control.
  • Ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. As used herein, "about” refers to the given value ⁇ 10%.
  • the subject is meant an individual.
  • the subject is a mammal such as a primate, and, more preferably, a human.
  • the "subject” can include domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.).
  • polypeptide is used synonymously herein with the term “peptide.” Both “polypeptide” and “peptide” include a series of naturally occurring or non-naturally occurring amino acids connected by peptide bonds.
  • isolated polypeptide or “purified polypeptide” is meant a polypeptide that is substantially free from the materials with which the polypeptide is normally associated in nature or in culture.
  • the polypeptides of the invention can be obtained, for example, by extraction from a natural source if available (for example, a mammalian cell), by expression of a recombinant nucleic acid encoding the polypeptide (for example, in a cell or in a cell-free translation system), or by chemically synthesizing the polypeptide.
  • polypeptide may be obtained by cleaving full length polypeptides. When the polypeptide is a fragment of a larger naturally occurring polypeptide, the isolated polypeptide is shorter than and excludes the full-length, naturally-occurring polypeptide of which it is a fragment.
  • the invention relates to novel polypeptide analogues of GLP-1 and exendin-4.
  • GLP-1 is used synonymously with GLP-1 7-36 amide, the amidated form of residues 7-36 of the complete GLP-1 sequence, and GLP-1 7-37.
  • Residues of exendin-4 are aligned with GLP-1, residues 7-36, and numbered according to the numbering of the GLP-1 residues. Such a residue numbering convention is used throughout. See Figure 1 .
  • the polypeptides in a preferred embodiment, are insulinotropic.
  • insulinotropic is meant that the polypeptides increase insulin synthesis, release or secretion in a glucose dependent manner as compared to levels of basal release in response to glucose alone.
  • Such increase in insulin release preferably is at least 1.15, 1.25, 1.5, 2.0, 2.5, 3.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, or 20.0 times greater than basal release.
  • the increase in insulin release can be shown directly (e.g., by showing increased levels of insulin) or indirectly (e.g., by showing reduced levels of glucose or by showing increased levels of cAMP) either in vivo (e.g., by assaying blood glucose levels) or in vitro (e.g., by assaying the level of insulin in the culture medium) using assay methods known in the art.
  • Insulinotropic effects can be due to any one of several mechanisms, including, for example, an increase in the number of insulin positive cells.
  • the insulinotropic polypeptides for example, promote insulin release by promoting differentiation of stem cells into insulin-positive cells and by promoting de-differentiation of non-stem cells to a less differentiated state and then promoting differentiation into insulin-positive cells.
  • the insulinotropic effects may be caused by an increase in the amount of insulin synthesized and/or released by each insulin positive cell in a given period of time. Combined insulinotropic effects could also occur if the number of insulin positive cells is increased and the amount of insulin secreted by each cell is also increased.
  • basic release is meant the amount of insulin released in response to a glucose stimulus in the absence of a second releasing agent.
  • insulin-positive cells any cells that have been shown to release insulin, including, for example, pancreatic islet cells, such as beta cells, or cell lines such as RIN 1048-36 cells, any cells designed to release insulin (e.g., genetically modified cells that contain insulin), or any cells that contain insulin.
  • analogue of GLP-1 or exendin-4" is meant modified GLP-1 and exendin amino acid sequences that show agonist properties (i.e., show one or more biological activities of GLP-1 or exendin-4).
  • modifications include chimeric polypeptides that include one or more amino acid residues present in GLP-1 and one or more amino acid residues present in exendin-4.
  • the modifications also include truncations of either GLP-1 or exendin-4 or the chimeric polypeptides. For example, a truncated chimeric polypeptide is exendin-4 7-36 with the G at position 36 replaced with the R in position 36 of GLP-1.
  • the polypeptides of the present invention include one or more additional amino acids (i.e., insertions or additions), deletions of amino acids, or substitutions in the amino acid sequence of GLP-1 or exendin-4 without appreciable loss of functional activity as compared to GLP-1 or exendin-4.
  • the deletion can consist of amino acids that are not essential to the presently defined differentiating activity and the substitution(s) can be conservative (i.e., basic, hydrophilic, or hydrophobic amino acids substituted for the same) or non-conservative.
  • modifications and changes may be made in the amino acid sequence of GLP-1 and exendin-4, and a protein having like characteristics still obtained.
  • Various changes may be made in the amino acid sequence of the GLP-1 or exendin-4 amino acid sequence (or underlying nucleic acid sequence) without appreciable loss of biological utility or activity and possibly with an increase in such utility or activity.
  • fragments or “truncations” as used herein regarding GLP-1 or exendin-4 or polypeptides having amino acid sequences substantially homologous thereto means a polypeptide sequence of at least 5 contiguous amino acids of either GLP-1, exendin 4, or polypeptides having amino acid sequences substantially homologous thereto, wherein the polypeptide sequence has an insulinotropic function.
  • D-enantiomers in which at least one naturally occurring L-configuration of an amino acid residue is replaced by the D-configuration of the amino acid residue.
  • the present invention contemplates the use of a spacer, such as a lateral spacer.
  • a spacer such as a lateral spacer.
  • the term "lateral spacer” is defined as a compound that is incorporated within the amino acid sequence by chemical bonds, whereby the compound increases the distance between two or more amino acid residues in order to reduce or eliminate the cleavage (e.g., by DPP 1V) of the amino acid sequence at or near that position.
  • cleavage of the sequence by an enzyme is reduced or eliminated when compared to the sequence in the absence of the lateral spacer (A-B).
  • 1 to 4 compounds can be incorporated into the amino acid sequence as the lateral spacer.
  • 1, 2, 3, or 4 compounds are inserted in various embodiments.
  • the lateral spacer is any compound that can form a peptide bond with an amino acid, i.e ., contains at least one amino group and at least one carboxyl group (C0 2 - ), where the carboxyl group can be a carboxylic acid or the ester or salt thereof.
  • the lateral spacer has the formula H 2 N-R 1 -CO 2 H (I), wherein R 1 comprises a substituted or unsubstituted, branched or straight chain C 1 to C 20 alkyl group, alkenyl group, or alkynyl group; a substituted or unsubstituted C 3 to C 8 cycloalkyl group; a substituted or unsubstituted C 6 to C 20 aryl group; or substituted or unsubstituted C 4 to C 20 heteroaryl group.
  • R 1 can be represented by the formula (CH 2 ) n , where n is from 1 to 10.
  • R 1 is (CH 2 ) 3 (3-aminopropionic acid) or (CH 2 ) 5 (6-aminohexanoic acid).
  • the present invention provides a purified polypeptide, wherein the polypeptide comprises a modified GLP-1 or exendin-4 sequence, or an anlogue thereof, with a spacer between the amino acid residues comparable to residues 7 and 8 (designated in the case of GLP-1 with a Aha spacer, for example, "GLP-1 Aha 8 ") or residues 8 and 9 (designated in the case of GLP-1 with a Aha spacer, for example, "GLP-1Aha 9 ”) of GLP-1.
  • the lateral spacer in one embodiment, is one or more aminoproprionic acid residues.
  • the spacer is a 6-aminohexanoic acid spacer and the 6-aminohexanoic acid spacer comprises less than four 6-aminohexanoic acid residues.
  • the polypeptide for example, can comprise GLP-1 7-36 with one or more 6-aminohexanoic acid residues between residues 7 and 8 (i.e., GLP-1 Aha 8 ) or can comprise GLP-1 7-36 with one or more 6-aminohexanoic acid residues between residues 8 and 9.
  • the polypeptide can comprise GLP-1 7-36 with two or more 6-aminohexanoic acid residues between residues 7 and 8 (i.e., GLP-1 Aha 8 ) or can comprise GLP-1 7-36 with two or more 6-aminohexanoic acid residues between residues 8 and 9.
  • the polypeptide for example, can comprise GLP-1 7-36 with three or more 6-aminohexanoic acid residues between residues 7 and 8 (i.e., GLP-1 Aha 8 ) or can comprise GLP-1 7-36 with three or more 6-aminohexanoic acid residues between residues 8 and 9.
  • polypeptide comprises the amino acid sequence of SEQ ID NO:8, SEQ ID NO:22, or SEQ ID NO:23.
  • polypeptide comprises the amino acid sequence of SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO: 48, or SEQ ID NO:49.
  • the polypeptide comprises the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:25, SEQ ID NO:33, wherein the amino acid sequence contains a spacer between the amino acid residues comparable to residues 7 and 8 or to residues 8 and 9 of GLP-1.
  • the polypeptide of the present invention has an insulinotropic effect that is comparable to the effect of an equimolar amount of GLP-1 or, in a more preferred embodiment, an insulinotropic effect that is comparable to the effect of an equimolar amount of exendin-4.
  • an insulinotropic effect is meant an effect that is within about 10-15% of the effect of GLP-1 or exendin-4.
  • the polypeptide has an insulinotropic effect that exceeds the insulinotropic effect of either GLP-1 or exendin-4.
  • exceeding the effect of GLP-1 or exendin-4 is meant an increase in insulinotropic effect compared to GLP-1 or exendin-4, preferably an increase that is greater than about 10% of the effect of GLP-1 or exendin-4.
  • the polypeptide of the present invention is as potent as GLP-1 or exendin-4, and in a more preferred embodiment is more potent that GLP-1 and, optionally, more potent than exendin-4.
  • the polypeptide of the present invention is longer acting than GLP-1.
  • the polypeptide is at least as long acting as exendin-4.
  • the polypeptide is longer acting than exendin-4.
  • longer acting is meant that the polypeptide is more resistant than GLP-1 or exendin-4 to at least one degradative enzyme.
  • the preferred embodiment of the polypeptide of the present invention is more resistant to degradation by the enzyme dipeptidyl dipeptidase (DPP1V) than is GLP-1 and, optionally, more resistant than exendin-4.
  • DPP1V dipeptidyl dipeptidase
  • Such resistance to one or more degradative enzymes can be assessed directly by detecting the amount of degradation products (e.g., the amount ofN-terminal degradation products) or the amount of un-cleaved polypeptide.
  • the resistance to one or more degradative enzymes can be detected indirectly by assessing the reduction in insulinotropic effect over time following administration of a polypeptide of the invention. For example, as the degradative enzymes cleave the polypeptides of the invention, plasma insulin levels should decline after a single administration. In a preferred embodiment this decline would be slower than for GLP-1 and perhaps even slower than for exendin-4.
  • the polypeptide has reduced antigenicity as compared to exendin-4. Antigenicity can be assessed using routine methods, such as biological assays designed to assess neutralizing antibodies and polypeptide clearance.
  • the polypeptide has a higher binding affinity for the GLP-1 receptor than the binding affinity of GLP-1 for the GLP-1 receptor. In a more preferred embodiment, the polypeptide has a higher binding affinity for the GLP-1 receptor than the binding affinity of exendin-4 for the GLP-1 receptor.
  • the polypeptide stimulates intracellular cAMP levels over basal levels more than GLP-1. In an even more preferred embodiment, the polypeptide stimulates intracellular cAMP levels over basal levels more than exendin-4.
  • the invention provides a purified polypeptide, the amino acid sequence of which comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:25, SEQ ID NO:33. More specifically, the invention provides a purified polypeptide, the amino acid sequence of which consists essentially of SEQ ID NO:5,
  • SEQ ID NO:6 SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:25, SEQ ID NO:33.
  • the invention provides a purified polypeptide, the amino acid sequence of which consists of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:25, SEQ ID NO:33.
  • the invention provides a purified polypeptide, the amino acid sequence of which comprises SEQ ID NO:3, 4, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, or 41. More specifically, the invention provides a purified polypeptide, the amino acid sequence of which consists essentially of SEQ ID NO:3, 4, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, or 41. Even more specifically, the invention provides a purified polypeptide, the amino acid sequence of which consists of SEQ ID NO:3, 4, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, or 41.
  • polypeptides of the invention can be prepared using any of a number of chemical polypeptide synthesis techniques well known to those of ordinary skill in the art including solution methods and solid phase methods.
  • Solid phase synthesis in which the C-terminal amino acid of the polypeptide sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is one synthetic method for preparing the polypeptides.
  • Techniques for solid phase synthesis are described by Merrifield et al., J. Am. Chem. Soc. 85:2149-2156 (1963 ). Many automated systems for performing solid phase peptide synthesis are commercially available.
  • Solid phase synthesis is started from the carboxy-terminal end (i.e., the C-terminus) of the polypeptide by coupling a protected amino acid via its carboxyl group to a suitable solid support.
  • the solid support used is not a critical feature provided that it is capable of binding to the carboxyl group while remaining substantially inert to the reagents utilized in the peptide synthesis procedure.
  • a starting material can be prepared by attaching an amino-protected amino acid via a benzyl ester linkage to a chloromethylated resin or a hydroxymethyl resin or via an amide bond to a benzhydrylamine (BHA) resin or p-methylbenzhydrylamine (MBHA) resin.
  • halomethyl resins such as chloromethyl resin or bromomethyl resin
  • hydroxymethyl resins such as phenol resins, such as 4-(a-[2,4-dimethoxyphenyl]-Fmoc-aminomethyl)phenoxy resin
  • tert-alkyloxycarbonyl-hydrazidated resins such as tert-alkyloxycarbonyl-hydrazidated resins
  • the acid form of the peptides may be prepared by the solid phase peptide synthesis procedure using a benzyl ester resin as a solid support.
  • the corresponding amides may be produced by using benzhydrylamine or methylbenzhydrylamine resin as the solid support.
  • the ⁇ -amino group of each amino acid used in the synthesis should be protected during the coupling reaction to prevent side reactions involving the reactive ⁇ -amino function.
  • Certain amino acids also contain reactive side-chain functional groups (e.g . sulfhydryl, amino, carboxyl, hydroxyl, etc: ) which must also be protected with appropriate protecting groups to prevent chemical reactions from occurring at those sites during the peptide synthesis.
  • Protecting groups are well known to those of skill in the art. See, for example, The Peptides: Analysis, Syntliesis, Biology, Vol. 3: Protection of Functional Groups in Peptide Synthesis (Gross and Meienhofer (eds.), Academic Press, N.Y. (1981 )).
  • a properly selected ⁇ -amino protecting group will render the ⁇ -amino function inert during the coupling reaction, will be readily removable after coupling under conditions that will not remove side chain protecting groups, will not alter the structure of the peptide fragment, and will prevent racemization upon activation immediately prior to coupling.
  • side-chain protecting groups must be chosen to render the side chain functional group inert during the synthesis, must be stable under the conditions used to remove the ⁇ -amino protecting group, and must be removable after completion of the peptide synthesis under conditions that will not alter the structure of the peptide.
  • Coupling of the amino acids may be accomplished by a variety of techniques known to those of skill in the art. Typical approaches involve either the conversion of the amino acid to a derivative that will render the carboxyl group more susceptible to reaction with the free N-terminal amino group of the peptide fragment, or use of a suitable coupling agent such as, for example, N,N'-dicyclohexylcarbodimide (DCC) or N,N'-diisopropylcarbodiimide (DIPCDI). Frequently, hydroxybenzotriazole (HOBt) is employed as a catalyst in these coupling reactions.
  • DCC N,N'-dicyclohexylcarbodimide
  • DIPCDI N,N'-diisopropylcarbodiimide
  • HOBt hydroxybenzotriazole
  • synthesis of the peptide is commenced by first coupling the C-terminal amino acid, which is protected at the N-amino position by a protecting group such as fluorenylmethyloxycarbonyl (Fmoc), to a solid support.
  • Fmoc-Asn Prior to coupling of Fmoc-Asn, the Fmoc residue has to be removed from the polymer.
  • Fmoc-Asn can, for example, be coupled to the 4-(a-[2,4-dimethoxyphenyl]-Fmoc-amino-methyl)phenoxy resin using N,N'-dicyclohexylcarbodimide (DCC) and hydroxybenzotriazole (HOBt) at about 25°C for about two hours with stirring.
  • DCC N,N'-dicyclohexylcarbodimide
  • HOBt hydroxybenzotriazole
  • the remaining Fmoc-protected amino acids are coupled stepwise in the desired order.
  • Appropriately protected amino acids are commercially available from a number of suppliers (e.g ., Novartis (Switzerland) or Bachem (Torrance, CA)).
  • appropriately protected peptide fragments consisting of more than one amino acid may also be coupled to the "growing" peptide. Selection of an appropriate coupling reagent, as explained above, is well known to those of skill in the art.
  • Each protected amino acid or amino acid sequence is introduced into the solid phase reactor in excess and the coupling is carried out in a medium of dimethylformamide (DMF), methylene chloride (CH 2 Cl 2 ), or mixtures thereof. If coupling is incomplete, the coupling reaction may be repeated before deprotection of the N-amino group and addition of the next amino acid. Coupling efficiency may be monitored by a number of means well known to those of skill in the art. A preferred method of monitoring coupling efficiency is by the ninhydrin reaction. Peptide synthesis reactions may be performed automatically using a number of commercially available peptide synthesizers such as the Biosearch 9500TM synthesizer (Biosearch, San Raphael, CA).
  • the peptide can be cleaved and the protecting groups removed by stirring the insoluble carrier or solid support in anhydrous, liquid hydrogen fluoride (HF) in the presence of anisole and dimethylsulfide at about 0°C for about 20 to 90 minutes, preferably 60 minutes; by bubbling hydrogen bromide (HBr) continuously through a 1 mg/10 mL suspension of the resin in trifluoroacetic acid (TFA) for 60 to 360 minutes at about room temperature, depending on the protecting groups selected; or by incubating the solid support inside the reaction column used for the solid phase synthesis with 90% trifluoroacetic acid, 5% water and 5% triethylsilane for about 30 to 60 minutes.
  • HF liquid hydrogen fluoride
  • TFA trifluoroacetic acid
  • Other deprotection methods well known to those of skill in the art may also be used.
  • the peptides can be isolated and purified from the reaction mixture by means of peptide purification well known to those of skill in the art.
  • the peptides may be purified using known chromatographic procedures such as reverse phase HPLC, gel permeation, ion exchange, size exclusion, affinity, partition, or countercurrent distribution.
  • polypeptides of the invention can also be prepared by other means including, for example, recombinant techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY , (Sambrook).
  • the invention further provides a method of treating a subject with diabetes, comprising administering to the subject the polypeptide of the invention in an amount that has an insulinotropic effect.
  • diabetes is meant diabetes mellitus.
  • the method of the present invention is considered to be useful in the treatment of a subject having type 2 diabetes.
  • the method of the present invention could be of use in other forms of diabetes (including, for example, type 1 diabetes) when the polypeptide promotes non-insulin producing cells to produce insulin.
  • the polypeptides of the present invention also have uses in the nervous system.
  • the polypeptides are neurotrophic (i.e. promoting proliferation, differentiation or neurite outgrowth) or neuroprotective (i.e. rescuing neuron cells or reducing neuronal cell death).
  • the invention further relates to a method of reducing neuronal death, comprising contacting one or more neurons with a polypeptide comprising GLP-1, exendin-4, or a neuroprotective or neurotrophic GLP-1 or exendin-4 analogue.
  • Neuronal death may occur, for example, with mechanical injury (e.g., trauma or surgery), toxic injury, neurodegenerative disease, apoptosis, and peripheral neuropathy.
  • rescuing neurons i.e., promoting viability of cells that show signs of cell death
  • reducing neuronal death i.e., promoting viability of cells that do not show signs of cell death
  • treatment with a compound that reduced neuronal death would be useful in treating an explant or culture of neuronal cells, prior to subsequent transplantation.
  • such treatment could be used to rescue neurons and reduce neuronal death following a stroke, brain or spinal cord injury, nerve injury, or neurotoxic injury.
  • rescuing neurons or reducing neuronal death would be useful in the treatment of neurodegenerative condition or disease diseases, including, for example, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, and peripheral neuropathy.
  • neurodegenerative condition or disease diseases including, for example, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, and peripheral neuropathy.
  • the invention also relates to a method of promoting neuronal differentiation or proliferation, comprising contacting one or more neurons or neuronal precursor cells with a polypeptide comprising GLP-1, exendin-4, or a differentiation-inducing or proliferation-inducing GLP-1 or exendin-4 analogue.
  • Differentiation involves a transition from a cell state in which the cell lacks neuronal characteristics (e.g., lacks characteristics such as a distinct nucleolus, neuronal processes, extensive rough endoplasmic reticulum, expression of neuronal markers) to a cell state characterized by a neuronal phenotype.
  • neuronal proliferation is meant that stem cells or cells of neuronal lineage divide and/or differentiate into neurons.
  • the effect of either differentiation or proliferation is an increase in the number of neurons.
  • an increase in the number of neurons is meant an addition of neurons to the total number of all neurons present.
  • the rate of neuronal cell death may exceed the rate of differentiation or proliferation, but the addition of new neurons is still considered to be an increase over the total neurons and such an increase in number, even in the absence of an increase in the total number of living neurons, could still have therapeutic advantages.
  • the present invention also relates to a method of reducing formation or accumulation of amyloid ⁇ protein, comprising contacting one or more neurons with a polypeptide comprising GLP-1, exendin-4, or a GLP-1 or exendin-4 analogue that affects ⁇ -amyloid precursor protein metabolism.
  • a method could be useful in lowering levels of amyloid protein or in preventing the deposition of amyloid protein, which is observed in senile plaques in a subject with Alzheimer's Disease.
  • the method of the present invention could reduce formation or accumulation of amyloid ⁇ protein by acting at various points in the processing of ⁇ -amyloid precursor protein.
  • the polypeptide may decrease synthesis of ⁇ -amyloid precursor protein, promote cleavage of ⁇ -amyloid precursor protein within the amyloid ⁇ protein region, increase secretion of soluble ⁇ -amyloid precursor protein, decrease secretion of amyloid ⁇ protein, or increase degradation of amyloid ⁇ protein.
  • the present invention also relates to a method of promoting growth of neuronal processes, comprising contacting one or more neurons with a polypeptide comprising GLP-1, exendin-4, or a process-promoting GLP-1 or exendin-4 analogue.
  • growth of neuronal processes is meant either an increase in the number of neuronal processes off of the soma, an increase in the complexity of neuronal processes (usually due to an increase in the number of branch points of axons or dendrites) or an increase in length of the processes.
  • the growth of neuronal processes may be desired in many contexts, including for example, following a peripheral nerve injury or an injury to the central nervous system where optimization of regenerative capacity is desired.
  • the existing neurons may be able to compensate for neruonal death with an enriched field of processes.
  • the present invention also relates to a method of treating a subject with a neurodegenerative condition or of reducing one or more symptoms of a neurodegenerative condition in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide comprising GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analogue. More specifically, the treatment could be directed to neurodegenerative conditions selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, stroke, multiple sclerosis, brain injury, spinal cord injury, and peripheral neuropathy.
  • a method of treating a subject with a neurotoxic injury or of reducing one or more symptoms of a neurotoxic injury in a subject comprising administering to the subject a therapeutically effective amount of a polypeptide comprising GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analogue.
  • a polypeptide comprising GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analogue.
  • Such administration could be before during or after the exposure to the neurotoxin.
  • Neurotoxins include the neurotoxic form of amyloid ⁇ -peptide, camptothecin, glutamate, etoposide, anti-cancer drugs, vinca alkaloids, 3-nitrognognonic acid, MPTP, domoic acid, kainic acid, and ibotenic acid.
  • the contacting step in these neural methods is performed in vivo or in vitro depending upon the desired effect.
  • neurons in culture can be treated prior to or after manipulation in culture that might cause neuronal death.
  • neurons in situ in the nervous system can be treated prior to or after exposure to a trigger that causes neuronal death.
  • the donor neurons to be transplanted might be treated in culture and then the transplantation area of the brain or spinal cord can be treated to prevent neuronal death of the recipient's neurons and of the transplanted neurons.
  • polypeptides related to uses in the nervous system include polypeptides comprising GLP-1, exendin-4, and their biologically active analogues or agonists.
  • the analogues bind and activate the GLP-1/exendin-4 receptor.
  • the polypeptides include for example polypeptides having the amino acid sequence of SEQ ID NO:1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 25, 33, 42, 43, 44, 45, 46, 47, or 48.
  • Other examples include polypeptides having the amino acid sequence of SEQ ID NO:3, 4, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, or 41.
  • compositions comprised of a polypeptide of the invention, including for example, GLP-1, exendin-4, and their biologically active analogues or agonists, in combination with a pharmaceutically acceptable carrier.
  • blood glucose levels could be monitored with normoglycemia being the optimal effect of treatment. If blood glucose levels are higher than preferred levels, then the amount of polypeptide administered should be increased, and, if blood glucose levels are lower than preferred levels, then the amount of polypeptide administered would be decreased.
  • the dosages of the polypeptides to be used in the in vivo method of the invention preferably range from about 0.1 pmoles/kg/minute to about 100 nmoles/ kg/minute for continuous administration and from about .01 nmoles/kg to about 400 nmoles/kg for bolus injection.
  • the dosage of the polypeptide in in vivo methods range from about 0.01 nmoles/kg/min to about 10 nmoles/kg/min.
  • the exact amount required will vary from polypeptide to polypeptide and subject to subject, depending on the species, age, and general condition of the subject, the severity of disease that is being treated, the particular polypeptide used, its mode of administration, and the like. Thus, it is not possible to specify an exact "insulinotropic amount" or an amount useful in treating neuronal disease or injury. However, an appropriate amount may be determined by one of ordinary skill in the art using only routine experimentation.
  • the polypeptides may be conveniently formulated into pharmaceutical compositions composed of one or more of the compounds in association with a pharmaceutically acceptable carrier.
  • the compounds may be administered orally, intravenously, intramuscularly, intraperitoneally, topically, transdermally, locally, systemically, intraventricularly, intracerebrally, subdurally, or intrathecally.
  • One skilled in the art would know to modify the mode of administration, the pharmacologic carrier, or other parameters to optimize the insulinotropic effects.
  • the amount of active compound administered will, of course, be dependent on the subject being treated, the subject's weight, the manner of administration and the judgment of the prescribing physician.
  • the pharmaceutical compositions may be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, lotions, creams, gels, or the like, preferably in unit dosage form suitable for single administration of a precise dosage.
  • the compositions will include, as noted above, an effective amount of the selected drug in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc. See, e.g., Remington's Pharmaceutical Sciences, latest edition, by E.W. Martin Mack Pub. Co., Easton, PA , which discloses typical carriers and conventional methods of preparing pharmaceutical compositions that may be used in conjunction with the preparation of formulations of the polypeptides and which is incorporated by reference herein.
  • conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc., an active compound as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
  • the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.
  • fine powders or granules may contain diluting, dispersing, and/or surface active agents, and may be presented in water or in a syrup, in capsules or sachets in the dry state, or in a nonaqueous solution or suspension wherein suspending agents may be included, in tablets wherein binders and lubricants may be included, or in a suspension in water or a syrup.
  • suspending agents may be included, in tablets wherein binders and lubricants may be included, or in a suspension in water or a syrup.
  • flavoring, preserving, suspending, thickening, or emulsifying agents may be included.
  • Tablets and granules are preferred oral administration forms, and these may be coated.
  • Parental administration if used, is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parental administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained. See, e.g., U.S. Patent No. 3,710,795 , which is incorporated by reference herein.
  • liquids, suspension, lotions, creams, gels or the like may be used as long as the active compound can be delivered to the surface of the skin.
  • a series of chimeric peptides was designed that incorporated fundamental features of exendin-4 and GLP-1.
  • the sequences of the 35 peptides are shown in Figure 1 , along with the sequence for GLP-1 (residues 7-36) and exendin-4 (residues 7-45 numbered according to the alignment of exendin-4 with the numbered GLP-1 residues). They were designed to (i) minimize the cleavage action of DDP1V between amino acids 8 and 9, (ii) assess the minimal requirement for insulinotropic action, and (iii) to assess which amino acid differences between exendin-4 and GLP-1 account for the former's 13-fold increase in potency versus GLP-1.
  • the peptides shown in Figure 1 utilized L- and D-amino acids in their synthesis.
  • Peptides were synthesized on a PEG-Polystyrene resin using Fmoc- derivatives of amino acids in a Applied Biosystems (Foster, CA) automated peptide synthesizer using piperidine-dimethyl formamide for deprotection and HOBt/HBTU for coupling.
  • the finished peptides were cleaved from the resin using trifluoroacetic acid (TFA), precipitated with ether and subjected to purification using reverse phase HPLC on a C-18 hydrophobic resin in 0.1%TFA using an acetonitrile gradient. The purity of the final material was verified using reverse phase HPLC and the mass of the peptide was verified using mass spectrometry. All peptides were of 95% or greater purity.
  • RIN 1048-36 cells a gift from Dr. Samuel A. Clark (Bio Hybrid Technologies, Shrewsbury, MA) were used to monitor the action of GLP-1, exendin-4 and analogs on insulin secretion.
  • Cells were seeded at a density of 2.5 x 10 5 cells/cm 2 on glass coverslips placed on the bottom of 12-well dishes and grown for 48 h. Thereafter, they were preincubated for 2 periods of 30 min each with glucose-free buffer (containing mM: 140 NaCl, 5 KCl, 1 NaPO4, 1 MgSO4, 2 CaCl, 20 HEPES buffer (pH 7.4), and 0.1% bovine serum albumin) in a 37°C humidified incubator.
  • glucose-free buffer containing mM: 140 NaCl, 5 KCl, 1 NaPO4, 1 MgSO4, 2 CaCl, 20 HEPES buffer (pH 7.4), and 0.1% bovine serum albumin
  • peptide 11 (SEQ ID NO:8) (having the 4 amino acid spacer before residue 8) potently induced insulin secretion whereas peptide 25 (SEQ ID NO: 22) (having a 4 amino acid spacer between residues 8 and 9) and peptide 26 (SEQ ID NO:23) (having an 8 amino acid spacer between residues 8 and 9) did not induce insulin secretion.
  • Replacement of amino acid 8 (alanine:A) in GLP-1 with a small neutral amino acid the peptide in the comparable position in exendin-4 (i.e., glycine:G) induced insulin secretion slightly more than exendin-4. See GLP-1 Gly 8 (SEQ ID NO:3).
  • peptide 8 (SEQ ID NO:5) (having an A ⁇ G substitution at position 8 and a V ⁇ L substitution at position 16), peptide 9 (SEQ ID NO: 6) (having A ⁇ G, V ⁇ L, S ⁇ K, Y ⁇ Q, and L ⁇ M substitutions at position 8, 16, 18, 19, and 20, respectively), peptide 10 (Ex-WOT; SEQ ID NO:7) (having the same substitutions as in peptide 9 and additional having G ⁇ E, Q ⁇ E, A ⁇ V, K ⁇ R, E ⁇ L, A ⁇ E, V ⁇ K, K ⁇ N, and R ⁇ G substitutions at residues 22, 23, 25, 26, 27, 30, 33, 34, 36, respectively) all retained the ability to induce insulin secretion.
  • peptide 8 (SEQ ID NO:5) had a substantially greater effect on insulin secretion than either GLP-1 or exen
  • Truncations of exendin-4 were also tested for their ability to induce insulin secretion. See peptides 15-24 (SEQ ID NOs:12-21). Only those peptides including more than 32 residues (i.e., peptides 15-18 (SEQ ID NOs:12-15)) induced insulin secretion. Of those truncation peptides that induced insulin secretion, peptide 15 (SEQ ID NO:12) (including residues up to and including residue 43) and peptide 18 (SEQ ID NO:15) (including residues up to and including residue 34) were the only ones that had an inducing effect that exceeded that of exendin-4 or GLP-1.
  • GLP-1 bears a net neutral charge, possessing a total of 4+ charges related to basic amino acids at positions 7, 26, 34 and 36, and 4- charges related to acidic amino acids at positions 9, 15, 21 and 27.
  • Exendin-4 bears a net negative charge related to a basic domain at position 21-23.
  • Exendin-4 possesses a total of 4+ charges (positions 7, 18, 26, 33) and 6- charges (positions 9, 15, 21, 22, 23, 30), whereas its 9 amino acid terminal tail is neutral.
  • exendin-4 a single basic amino acid (providing a positive charge) for the tail of exendin-4 (i.e., the replacement of small neutral glycine, G, by larger arginine, R, in position 36), as in peptide 34 (SEQ ID NO:31), results in inactivity of the peptide in in vitro insulin secretion.
  • Arginine, R is well tolerated in position 36 of GLP-1, and when retained or replaced in GLP-1 by neutral glycine, G, and the exendin-4 tail (peptide 12(SEQ ID NO:9) it remains active and actually has an activity that exceeds that of exendin-4.
  • arginine, R is well tolerated in position 36 of exendin-4 when position 30 is modified from acidic glutamic acid, E, to neutral alanine, A (peptide 36 (SEQ ID NO:33)) when position 27 bears a negative charge.
  • GLP-1 Aha 8 (peptide 11; SEQ ID NO:8) was shown to be as effective as GLP-1.
  • GLP-1 Aha 8 (peptide 11; SEQ ID NO:8) induced insulin secretion about 1.2-fold above basal levels. See Figure 3 .
  • CHO cells stably transfected with the human GLP-1 receptor, GLP-1R cells were grown to 60-70% confluency on 12-well plates, washed three times with Krebs-Ringer phosphate buffer (KRP), and incubated with 1ml of KRP containing 0.1% bovine serum albumin (BSA) for 2h at 37°C in a humidified air incubator. Cells were then incubated in 1ml of KRP supplemented with 0.1% BSA with Isobutylmethylxanthine (IBMX) (1mM; Calbiochem, La Jolla, CA) in the presence or absence of the peptides under study.
  • KRP Krebs-Ringer phosphate buffer
  • BSA bovine serum albumin
  • IBMX Isobutylmethylxanthine
  • the reaction was stopped 30 min later by washing the intact cells three times with ice-cold phosphate buffered saline (PBS).
  • the intracellular cAMP was extracted by incubating the cells in ice-cold perchloric acid (0.6M, 1ml, 5 min). After adjusting the pH of the samples to 7 using potassium carbonate (5M, 84 ⁇ l), sample tubes were vortexed and the precipitate formed was sedimented by centrifugation (5 min, 2000 x g, 4°C). The supernatant was vacuum-dried and solubilized in 0.05M Tris (pH 7.5) containing 4mM EDTA, (300 ⁇ l).
  • cAMP levels of cAMP were measured in cells treated with GLP-1, the 6-aminohexanoic acid-containing peptides, or the D-amino acid containing peptides. Intracellular cAMP levels generated by the GLP-1 analogs were assessed initially at a peptide concentration of 10nM (the concentration at which maximum cAMP production is seen with GLP-1). The data are shown in Figure 4 . The peptides were incubated with the CHO/GLP-1 R cells in the presence of IBMX for 30 min at 37°C.
  • Table 3 shows the ED50 values of all three compounds.
  • GLP-1 Aha 8 (0.5nM) stimulated intracellular cAMP production to 4-fold above basal it however exhibited a higher ED50 when compared to GLP-1 and GLP-1 Gly 8 .
  • Binding studies were performed in the manner of Montrose-Rafizadeh et al. (1997b), J. Biol. Chem. 272:21201-206 . Briefly CHO/GLP-1R cells were grown to confluency on 12-well plates and washed with serum-free Ham F-12 medium for 2 h before the experiment. After two washes in 0.5ml binding buffer (10), cells were incubated overnight at 4 C with 0.5ml buffer containing 2% BSA, 17mg/L Diprotin A (Bachem, Torence, CA), 10mM glucose, 1-1000nM GLP-1 or other peptides and 30,000cpm SI-GLP-1 (Amersham, Philadelphia, PA).
  • the time-dependent duration of insulinotropic action was evaluated by quantifying plasma insulin and glucose levels in Zucker rats following intraperitoneal (i.p.) peptide administration. Specifically, after overnight fasting, diabetic male rats, approximately 400 g weight, were anesthetized with 50 mg/kg pentobarbital and a catheter was tied into their right femoral artery for blood collection. Thereafter, a bolus of exendin-4, GLP-1 or peptide (0.4 nmol/kg) was administered i.p. (N ⁇ 2 per peptide).
  • Plasma samples taken prior to peptide administration, at 30 and 60 min, and at 2, 4, 6 and 24 h, was drawn into heparinized tubes containing EDTA and aprotinin for insulin determination, and a separate sample was taken to measure glucose. Plasma was separated, removed and immediately frozen to -70°C. Thereafter insulin levels were quantified by using a rat insulin ELISA kit (Crystal Chem Inc., Chicago, IL) and plasma glucose was quantified by the glucose oxidase method.
  • polypeptide 10 As shown in Figure 8 , specific amino acids modifications provide a long duration of action on in vivo insulin.
  • the action of polypeptide 10 (Ex-4 WOT; SEQ ID NO:7) on plasma insulin levels proved to be long acting, like exendin-4.
  • polypeptide 10 proved to be more potent than equimolar exendin-4 in diabetic rats.
  • polypeptide 1 GLP-1 Gly 8 ; SEQ ID NO:3
  • polypeptide 11 GLP-1 Aha 8 ; SEQ ID NO:8 proved to have an action on the time-dependent insulin response that was intermediate between GLP-1 and exendin-4; being longer than the former but shorter than the latter.
  • polypeptides 1 and 11 proved to be more potent than equimolar GLP-1 in diabetic rats.
  • GLP-1 (2 ⁇ M) and GLP-1 Aha 8 (SEQ ID NO:8) (2 ⁇ M) were incubated with 5mU recombinant DPP1V (Calbiochem, La Jolla, CA) in PBS for 10 min and 2 h respectively at 37°C. Both compounds (100 ⁇ M, 100 ⁇ l) were incubated in an equivalent volume of human serum at 37°C for 2 h. In all cases, enzymatic reactions were quenched by the addition of trifluoroacetic acid (0.1% v/v final concentration). Samples were immediately analyzed using Matrix Assisted Linear Desorption Ionisation - Time Of Flight (MALDI-TOF) mass spectrometry.
  • MALDI-TOF Matrix Assisted Linear Desorption Ionisation - Time Of Flight
  • a Micromass MALDI-TOF (Micromass, Beverly, MA) reflectron instrument was used at a laser energy of 15-25% over a mass range of 1000 - 6000 Da, with 5 laser shots summed per spectrum.
  • Alpha-cyano-4-hydroxycinnamicacid (Sigma, St. Louis, MO) was used as a matrix and was prepared to a concentration of 10 mg/ml in a 8 mg/ml ammonium carbonate (Sigma, St.Louis, MO) buffer.
  • One microlitre samples were diluted 50/50 v/v with matrix before being transferred to the MALDI plate.
  • GLP-1 Aha 8 The stability of GLP-1 Aha 8 (SEQ ID NO:8) was compared with GLP-1 in the presence of DPP1V and human serum.
  • Treatment of GLP-1 (2 ⁇ M) with DPP1V (5mU) for 10 min or 100% serum for 2 h at 37°C caused a considerable increase in the amount of N-terminal truncated product (Mr 3089 gmol-1) as measured by MALDI.
  • Mr 3089 gmol-1
  • IBMX Isobutylmethylxanthine
  • Exendin-4 and GLP-1-(7-36)amide were obtained from Bachem (Torrance, CA).
  • the cloned rat insulinoma cell line RIN 1046-38 was a gift from Dr. Samuel A.
  • Clark Bio Hybrid Technologies, Shrewsbury, MA
  • mice were routinely cultured in M199 with Earle's salts (Mediatech, Inc., Herndon, VA) suppplemented with glucose (11mM), 50U/ml penicillin, 50 ⁇ g/ml streptomycin, and glutamine (2mM) in a humidified 5%CO 2 -95 air incubator at 37 C.
  • Chinese hamster ovary (CHO) cells stably transfected with the human GLP-1 receptor, CHO/GLP-1R cells, were described above.
  • Plasma insulin levels were measured by ELISA (Crystal Chem Inc., Chicago ILL).
  • HbA1c was measured as described in Greig et al., 1999. Blood glucose levels were measured using a Glucometer Elite (Bayer Diagnostics, Tarrytown, NY).
  • the underlined amino acids refer to those from the exendin-4 sequence that are being studied.
  • RIN 1046-38 cells grown to 60-70% confluence on 12-well plates were treated as described in Example 3 and cAMP determinations were performed accordingly. Dose response curves are shown in Figure 21 .
  • Table 5 shows IC 50 and EC 50 values derived from the competitive binding in CHO GLP-1R cells and cAMP assays in RIN 1046-38 cells respectively.
  • Table 5 The IC 50 and EC 50 values derived from the competitive binding in CHO GLP-1R cells and cAMP assays in RIN 1046-38 cells respectively.
  • the concentration that reached 50% of [ I 125 ] GLP-1 binding was calculated in three or four separate experiments performed in triplicate. Peptides of a few as 11 amino acids were assessed and such amino acids had minimal binding.
  • mice were housed in our facilities for 2.5 months to facilitate their acclimatization before the experiment commenced.
  • the animals were given Ex (1-30) (1nmol/kg) by intraperitoneal (ip) injection daily at 9am. Thereafter, animals received ip Ex (1-30) (1nmol/kg) at approximately 9am and 9pm for the following 32 days.
  • blood glucose levels and HbA1c were measured.
  • Food intake and animal weight was determined daily at the time of the 9am injection. No day was missed in the schedule.
  • Magnetic resonance images (MRI) were taken on 51 and an IPGTT was performed on day 52.
  • the HbA1c were 7.8 ⁇ 0.4 for the Ex(1-30) treated mice and 7.7 ⁇ 0.3 for the saline treated mice.
  • the fasting blood glucose values were 412 ⁇ 92 mg/dl for the Ex(1-30) treated mice and 600 ⁇ 1mg/dl for the saline treated mice.
  • Magnetic resonance images were obtained using a 1.9T, 31cm bore Bruker BioSpec system (Bruker Medizintechnik GmbH, Ettlingen, Germany), a 20cm inner diameter shielded gradient set and a 5cm diameter volume resonator.
  • the field of view was 5 x 5 cm over 128 x 128 pixels.
  • Each image was acquired using 8 acquisitions, over a total imaging time of approximately 9 minutes. Imaging was performed on all animals at two time points (day 0 and day 51).
  • a ⁇ amyloid- ⁇ peptide
  • ⁇ APP glycosylated membrane-bound protein ⁇ -amyloid precursor protein
  • sAPP soluble derivative
  • PC 12 cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated horse serum, 5% heat-inactivated fetal calf serum, 25mM Hepes buffer and 1x antibiotic-antimycotic solution (all culture media and sera were obtained from MediaTech Inc. (Herndon VA). Treatments were carried out in low serum in the presence of GLP-1 (3.3, 33, and 330 ⁇ g/ml) (Bachem, Torrance, CA), and two analogues, exendin-4 (0.1, 1.0 and 10 ⁇ g/ml) and exendin-4-WOT (peptide 10 (Ex-4 WOT; SEQ ID NO:7)) (0.1 and 1.0 ⁇ g/ml).
  • GLP-1 3.3, 33, and 330 ⁇ g/ml
  • exendin-4-WOT peptide 10 (Ex-4 WOT; SEQ ID NO:7)
  • NGF 10, 10, 25 and 50ng/ml
  • PC12 cells were used as a positive control.
  • conditioned media and cell lysates from untreated (low serum medium alone) and treated cells were subjected to immunoblot analysis using the monoclonal antibody, 22C11 (Roche Molecular Biochemicals, Indianapolis, IN). The antibody, raised against E.
  • Coli-made ⁇ APP whose epitope region has been assigned to ⁇ APP 66-81 in the ectoplasmic cysteine-containing domain, recognizes all mature forms of ⁇ APP found in cell membranes, as well as carboxy-truncated soluble forms secreted into conditioned media.
  • M r 100-140 kDa high molecular weight protein bands
  • LDH lactate dehydrogenase
  • the data indicate reduced levels of secreted derivatives and mature forms of ⁇ APP following GLP-1 treatment in PC12 cells. These reductions in sAPP secretion may be a consequence of reduced ⁇ APP synthesis.
  • GLP-1 and two of its long-acting analogues, exendin-4 and exendin-4 WOT, on neuronal proliferation and differentiation and on the metabolism of neuronal proteins in the rat pheochromocytoma (PC12) cell line were tested.
  • GLP-1 and exendin-4 induced neurite outgrowth, which was reversed by co-incubation with the selective GLP-1 receptor antagonist, exendin (9-39).
  • exendin-4 enhanced nerve growth factor (NGF) initiated differentiation and rescued degenerating cells following NGF-mediated withdrawal.
  • NGF nerve growth factor
  • Pheochromocytoma cells were obtained from Dr. D.K. Lahiri (Indianapolis) and RIN 1046-38 cells (a clonal rat insulinoma cell line) were a gift from Dr. Samuel A. Clark (Bio Hybrid Technology, Shrewsbury, MA).
  • PC12 cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated horse serum, 5% heat-inactivated fetal calf serum, 25mM Hepes buffer and 1x antibiotic-antimycotic solution.
  • RIN 1046-38 cells were grown in medium 199 containing 12mM glucose and supplemented with 5% heat-inactivated fetal calf serum, 0.03% glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin.
  • Cell culture media and sera were obtained from MediaTech (Cellgro), Inc.(Herndon, VA). The cells were grown in a humidified atmosphere containing 5.0% CO 2 . They were seeded at approximately 2.0x10 6 cells per 60mm dish.
  • PC12 cells were grown on cultureware coated in rat-tail collagen (Roche Molecular Biochemicals, Indianapolis). The 7S NGF was prepared by dilution in growth media at a concentration of 100 mg/ml and stored at -20°C. Stock solutions of GLP-1 and analogues were made fresh in sterile water and stored at -20°C.
  • Treatments began 24 hours after seeding, once cells were well attached. The medium was aspirated, and 3ml of fresh low serum media (containing only 0.5% fetal calf serum) with the appropriate compound(s), added.
  • Cell lysates were prepared as follows. The cells from the plate were collected gently and centrifuged at 800g for 10 minutes. The cell pellet was suspended in lysis buffer containing 10mM Tris-HCl (pH 7.4), 1% SDS, 0.174mg/ml phenylmethylsulfonyl fluoride (PMSF), 1mg/ml each of aprotinin, leupeptin, pepstatin A, and 4ml of a mixture of 45.98mg/ml sodium vanadate and 10.5mg/ml sodium fluoride. The suspended cells were triturated and centrifuged at 14,000g for 15 minutes. The proteins of the supernatant solution (cell lysate) were measured (Bradford, 1976) and analysed by immunoblotting.
  • PMSF phenylmethylsulfonyl fluoride
  • PC12 cells were grown on 60 mm dishes as above and cultured for four days. During this time neurite outgrowth was quantified daily. Five random fields of cells were evaluated per dish and the proportion of neurite-bearing cells was determined. Approximately 100 cells per field were scored for neurites equal to or greater in length than that of the cell body. A cell was only scored once, although it may have had more than one process per cell.
  • PC12 cells when grown in complete media without the presence of neurotrophic compounds, displayed none of the characteristics of neuronal cell types. When exposed to NGF in low serum medium, the cells stopped dividing and developed morphological properties similar to sympathetic neurons. The cells extended long processes, some becoming highly branched with the cell body exhibiting a more flattened appearance than in cells cultured in low serum medium alone.
  • GLP-1 or exendin-4 Treatment with GLP-1 or exendin-4 in low serum medium produced similar effects on differentiation to those induced by NGF. GLP-1 and exendin-4 induced neurites were generally shorter in length, and less branched than neurites generated following treatment with NGF. In contrast, the GLP-1 antagonist, exendin (9-39) in combination with exendin-4 failed to initiate neurite extension.
  • PC12 cells were grown in complete media (RPMI 1640 + 5% Fetal bovine serum + 10% Horse serum) in the presence / absence of 50 ng/ml NGF or the presence/ absence of exendin-4 (1 or 5 mg/ml). Cells were harvested after 4 or 7 days, and subsequently allowed to rejuvenate in regular media for an additional 3 days. On the final day, cells were harvested and a MTT assay was performed to determine the proportion of viable cells. In a second series of experiments (prevention) cells were cultured in the presence of 50 ng/ml NGF and exendin-4 (1 or 5 mg/ml) for 4 or 7 days. Cells were harvested and allowed to rejuvenate as above.
  • the CellTiter 96® Aqueous One Solution Cell Proliferation Assay Reagent from Promega (Madison, WI) was used in a colourimetric procedure for determination of the number of viable cells in a modified MTT assay.
  • the Reagent contains a novel tetrazolium compound [3-(4,5-dimethyl-2-yl)-5-(3 carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES).
  • PES has enhanced chemical stability, which allows combination with MTS to form a stable solution.
  • the MTS tetrazolium compound is bioreduced by cells into a coloured formazan product, which is soluble in tissue culture medium. This conversion is presumably accomplished by NADPH or NADH produced by dehydrogenase enzymes in metabolically active cells (Berridge and Tan 1993). Assays are performed by the addition of a small amount of the Reagent directly to cultured wells, incubation for 1-4 hours and subsequent absorbance at 490 nm with a 96-well plate reader. The quantity of formazan product, as measured by the amount of 490nm absorbance, is directly proportional to the number of living cells in culture. Since the MTS formazan product is soluble in tissue culture medium the present procedure requires fewer steps than procedures that use tetrazolium components such as MTT.
  • the cultures were treated for 48 hours with the GLP-1 antagonist, exendin (9-39); with the PI3 kinase inhibitor, LY294002 (40 ⁇ M); with the MAP kinase inhibitor, PD98059 (50 ⁇ M) or with the PKA inhibitor, H89 (20 ⁇ M).
  • Cells were seeded onto 60 mm dishes at approximately 1x10 5 cells/ml and treated with either 10nM GLP-1 or 0.3 ⁇ M exendin-4 with each of the aforementioned compounds.
  • NGF at 50 ng/ml and forskolin (PKA activator) (20 ⁇ M) were used as positive controls in these treatments.
  • Both PD98059 and LY294002 reduced GLP-1 and exendin-4 induced neurite outgrowth of the cells. Similarly, NGF-induced neuritic extension was reduced following PD98059 and LY294002 treatment. The involvement of both the ERK MAP kinase and the PI3 kinase signaling pathways is thus implicated in GLP-1 and exendin-4 mediated neurite production in PC12 cells. Treatment with H89 demonstrated some inhibitory effects on GLP-1 and NGF-induced neurite outgrowth. These data suggest that PKA is involved in the regulation of the MAP kinase signaling pathway but other signaling pathways are also involved.
  • synaptophysin which is a 37 kDa phosphorylated protein well expressed in the synaptic vesicle membrane.
  • the synaptophysin monoclonal antibody (Oncogene Research Products, San Diego CA), which stains neurosecretory vesicles of PC12 cells, was used.
  • the membranes were blocked with 20mM Tris, 500mM NaCl pH 7.4, 1% (w/v) casein (BioRad, San Diego CA) at 37°C for 1 hour. Primary antibody was diluted in block and incubated with the proteins overnight at 4°C.
  • the membrane was vigorously washed with 20mM Tris pH 7.4, 150mM NaCl and 0.05% Tween-20 (TBST), three times for 15 minutes at room temperature.
  • the peroxidase-linked secondary antibody in block was incubated with the membrane for 2 hours at room temperature.
  • Peroxidase-linked anti-mouse IgM (Chemicon, Tenecula, CA) was used as the secondary antibody against synaptophysin. Excess antibody was washed off with three vigorous washes in TBST prior to incubation in ECL Plus (Amersham, Philadelphia, PA) for 5 minutes.
  • the membrane was subsequently exposed to photographic film. Densitometric quantification of the protein bands was performed using Molecular Analyst software (BioRad, Hercules, CA).
  • Beta-2/NeuroD plays a major role in both neuronal and pancreatic endocrine development. Expression of NeuroD appears to be transient in sensory and motor neurons of the peripheral nervous system, sensory organs as well as parts of the brain and spinal cord during neuronal differentiation; however detection in the adult brain may suggest a secondary role in mature neurons (Lee et al 1997). Beta-2 expression in pancreatic endocrine cells, the intestine and the brain, activates insulin gene transcription and can induce neurons to differentiate.
  • Beta-2/NeuroD is essential for in vivo pancreatic development and neuronal differentiation.
  • NeuroD production was determined by Western blot analysis using anti-NeuroD antibody as described above for synaptophysin antibody, except peroxidase-linked anti-goat IgG (Santa Cruz Biotechnology Inc.) was used as the secondary antibody.
  • Beta-2/NeuroD expression is increased following treatment with GLP-1, providing further evidence for the neuronal differentiation properties of this insulinotropic polypeptide. As anticipated, cultures exposed to low serum medium alone showed nominal expression of Beta-2/NeuroD.
  • PC12 cells were plated onto poly-L-lysine coated glass coverslips in 35mm culture dishes and grown under standard conditions (as described above). Cells were fixed with 0.25% glutaraldehyde for 30 minutes. Endogenous peroxidase activity was quenched with 0.3% H 2 O 2 and incubation in the primary polyclonal antibody (dilution factor 1:1500) raised against the N-terminal of the GLP-1 receptor (a gift from Dr. Joel F. Habener, Massachusetts General Hospital, MA) was carried out at room temperature for 1 hour. Visualization used the avidin-biotin peroxidase method with subsequent development in diaminobenzidine dihydrochloride (DAB) following incubation in the biotinylated anti-rabbit IgG secondary antibody.
  • DAB diaminobenzidine dihydrochloride
  • GLP-1R-positive immunoreactive staining confirmed the presence of the GLP-1 receptor. More specifically, staining was on the cell body and to a lesser extent on the neurite terminal. However, not all PC12 cells expressed positive immunoreactive staining to the same degree, although almost all cells appeared positive.
  • RT-PCR Reverse transcriptase polymerase chain reaction
  • RT-PCR was performed as a sensitive assay for GLP-1 receptor mRNA.
  • Rat insulinoma cells (RIN cells) were used as a positive control.
  • Total RNA was isolated from PC12 cells using the method of Chomczynski and Sacchi (1987). 2.5mg RNA was used in our RT-PCR reaction.
  • RT-PCR was undertaken in a volume of 50ml of buffer containing 50mM KC1, 10mM Tris-HCl, 3.5mM MgC12), 200mM dNTP's and 0.4mM of each rat GLP-1R sense (5' ACAGGTCTCTTCTGCAACC 3') and antisense (5' AAGATGACTTCATGCGTGCC 3') oligonucleotide primers (5'- and 3'- ends of the pancreatic GLP-1 receptor sequences). Amplification was undertaken for 30 cycles in the presence of [a-32P]dCTP. Rat islet cells were used as the positive control. RT-PCR products (10ml) were separated on a 4-20% polyacrylamide gel with appropriate size markers. The gel was subsequently dried under a vacuum at 80°C for 1 hour and exposed to X-ray film.
  • RT-PCR products of the expected size for the GLP-1 receptor were obtained. Clear bands at 928-bp in rat islet mRNA and PC12 cell mRNA confirmed the presence of the GLP-1 receptor on PC12 cells.
  • PC12 cells were treated with 33 ⁇ g/ml GLP-1 for 3 days. Triplicate cultures were harvested at 5-minute intervals after the onset of treatment for a total period of 30 minutes. Cells harvested at the start of treatment (zero minutes) were used for baseline levels of cAMP. Cyclic AMP was measured according to the method of Montrose-Rafizadeh et al (1997a).
  • LDH assay The potentially toxic effects of exendin-4 were tested in vitro by two methods: LDH assay and trypan blue exclusion method.
  • the LDH assay was performed using a Sigma kit. Conditioned media samples collected at different time intervals following treatments were subjected to a sensitive lactate dehydrogenase (LDH) assay.
  • LDH assay provided a measure of the number of cells via total cytoplasmic LDH or by membrane integrity as a function of the amount of cytoplasmic LDH released into the medium.
  • the measurement of released LDH was based on the reduction of NAD by the action of LDH.
  • the resulting reduced NAD (NADH) was utilized in the stoichiometric conversion of a tetrazolium dye.
  • the final coloured compound was measured spectrophotometrically. If the cells were lysed prior to assaying the medium, an increase or decrease in cell number resulted in a concomitant change in the amount of substrate converted. This indicated the degree of cytolysis or membrane damage (cytotoxicity) caused by the test material.
  • Proliferating cells (those that were undergoing DNA replication at the time of BrdU labeling) exhibited dark-staining nuclei with the chromagen reaction. BrdU incorporation was quantitated on days 1, 2 and 3 of treatment. Three dishes for each treatment condition were counted and expressed as the percentage of labeled cells relative to the total number of cells. PC12 cells showed increased incorporation of BrdU on day 1 following treatment with NGF (9% increase relative to untreated) and GLP-1 (18% relative to untreated).
  • GLP-1 and its long-acting analogue, exendin-4 to protect cultured hippocampal neurons against cell death induced by glutamate, and to attenuate ibotenic acid-induced cholinergic marker deficit in adult rats was tested.
  • Hippocampal neuronal cultures were prepared from 18-day-old embryonic Sprague Dawley rats using methods similar to those described previously (Mattson et al., 1995). Briefly, cells were dissociated by mild trypsination and trituration and plated in Minimal Essential Medium containing 10% FBS and 1% antibiotic solution (10 4 U/ml penicillin G, 10 mg/ml streptomycin and 25 ⁇ g/ml amphotericin B; Sigma Chemicals, St. Louis, MO). Hippocampal neurons were plated at a density of 100,000 cells/ml on 25 mm diameter poly-D-lysine coated glass coverslips. Three hours after plating the media was replaced with serum-free Neurobasal medium containing 1% B-27 supplement (Gibco/Life Technologies, Carlsbad, CA).
  • MAP-2 neurotrophic factor-2
  • GFAP astrocytes
  • Binding studies were performed as described by Montrose-Rafizadeh (1997b). Duplicate hippocampal neuronal cultures were washed in 0.5ml binding buffer and subsequently incubated in 0.5ml buffer containing 2% BSA, 17 mg/liter diprotin A (Bachem, Torrance, CA), 10mM glucose, 0.001-1000 nM GLP-1 and 30,000 cpm 125 I-GLP-1 (Amersham Pharmacia Biotech, Little Chalfont, UK), overnight at 4°C. At the end of the incubation the supernatant was discarded, and the cells washed three times in ice-cold PBS and incubated at room temperature with 0.5ml 0.5M NaOH and 0.1% SDS for 10 min.
  • Radioactivity in cell lysates was measured in an Apec-Series ⁇ -counter (ICN Biomedicals, Inc., Costa Mesa, CA). Specific binding was determined as the total binding minus the radioactivity associated with cells incubated in the presence of a large excess ofunlabelled GLP-1 (1 ⁇ M). The GLP-1 concentration associated with 50% binding, EC 50 , was determined by logit plot analysis.
  • Binding of 125 I-GLP-1 to cultured hippocampal neurons was displaced, concentration-dependently, by unlabelled GLP-1 ( Fig 16A ).
  • cyclic AMP was measured according to the method of Montrose-Rafizadeh et al., (1997a). Triplicate hippocampal neuronal cell cultures were treated with 10 nM GLP-1 and harvested at 5-minute intervals after the onset of drug treatment for a total period of 30 minutes. Cells harvested at the start of drug treatment (zero minutes) were used for baseline levels of cAMP.
  • the fluorescent DNA binding dye Hoescht 33342 was used to measure apoptotic cell death. Neurons were incubated in Locke's buffer with GLP-1 (10 nM) or exendin-4 (0.3 ⁇ M) in the presence of absence of glutamate (10 ⁇ M) for 16 h. The concentration of GLP-1 used was based on the EC 50 value derived from the binding experiment, which was demonstrated to stimulate the release of cAMP, and which induced differentiation without causing cell death in our previous neuronal cell studies. Cells were fixed in a solution of 4% paraformaldehyde in PBS and membranes were permeabilized with 0.2% Trition X-100.
  • nuclei were visualized under epifluorescence illumination (340 nm excitation, 510 nm barrier filter) using a 40x oil-immersion objective. Approximately 200 cells were counted in at least three separate dishes for each treatment condition, and experiments were repeated at least twice. Cells were considered apoptotic if nuclear DNA was fragmented or condensed, whereas cells with nuclear DNA of a more diffuse and uniform distribution, were considered viable. At the time of counting, the investigator was unaware of the identity of the treatment groups. The percentage of cells with condensed or fragmented nuclei was determined in each culture.
  • the values represent the pooled means of six individual experiments.
  • the percentage of cells undergoing apoptosis as a result of each treatment condition were subjected to ANOVA using StatView statistical software (Cary, NC). Following significant main effects, a posteriori comparisons of treatment vs. control were made using Tukey's Honestly Significant Difference (HSD) test, using the pooled ANOVA error term and degrees of freedom.
  • HSD Tukey's Honestly Significant Difference
  • the cannulae were attached via a catheter to an osmotic minipump (ALZA Pharmaceuticals, Mountain View, CA). Pumps were filled with 2 x 10 -8 M GLP-1, 2 x 10 -9 M exendin-4 or vehicle (artificial cerebrospinal fluid). Both peptides were diluted in vehicle.
  • the pumps were set to deliver 0.25 ⁇ l/h over 14 days (total of 5.54 ng GLP-1 at 0.8 nM / kg / min and 0.7 ng exendin-4 at 0.08 nM / kg / min).
  • the brain infusion kits were assembled 5-6 h prior to implantation, and left in sterile saline at 37°C.
  • the minipumps were inserted into a subcutaneous pocket between the shoulder blades, the wounds sutured and the animals allowed to recover. Animals receiving infusions of GLP-1 or exendin-4, became modestly aphagic and adipsic, as a result of the insulinotropic nature of the peptides, which resulted in a slight drop in body weight. This was recouped within 3-4 days with the administration of twice daily fluids (0.9% saline) and soft diet, and by the time of sacrifice there were no differences in body weight between the groups.
  • mice On expiry of the minipumps (14 days), animals were terminally anaesthetized 0.1mg/kg sodium pentobarbitone and transcardially perfused with 100-150ml PBS (pH 7.4) followed by 250-350ml 4% paraformaldehyde solution in PBS, at a constant pressure of 100 mm Hg over a period of 15-20 min.
  • the brains were taken for immunocytochemical assessment and quantification of the lesion-induced damage and any resulting effects of peptide infusion on the cholinergic contingent of the basal forebrain.
  • Adjacent coronal brain sections were taken at 40 ⁇ m thickness, through the lesion area, and processed free-floating for ChAT; using the polyclonal goat anti-ChAT antibody at 1:100 dilution (Chemicon, International Inc., Temecula, CA), and glial fibrillary acidic protein (GFAP); using the polyclonal rabbit anti-GFAP antibody at 1:750 dilution (Chemicon). Visualization of positive immunoreactivity was carried out using an avidin-biotin/horse radish peroxidase protocol.
  • ChAT-positive immunoreactive cell bodies in the forebrain area were visualized under x100 magnification and manually counted on both sides.
  • the raw counts were corrected with the Abercrombie (1946) formula for an estimate of the total number of cell bodies in the area. Characterization of the cell loss in the basal nucleus as a result of the ibotenic acid lesion, was made by comparison of left (lesion side) relative to right (infusion side) counts, and the data presented as the percent change.
  • the animal names were coded such that cell counts were formally conducted blind to the experimental condition. Differences were subjected to analysis of variance, and a posteriori comparisons using Tukey's HSD test as above. Significance was accepted at p ⁇ 0.05 for all statistical analyses.
  • Choline acetyltransferase immunoreactivity was used as a marker for cholinergic neurons throughout the basal forebrain.
  • the ChAT antibody stained numerous large multipolar neurons, with a similar size and distribution to the acetylcholinesterase (AChE) positive cells.
  • the immunocytochemical staining had low background and provided a clear picture of the cell morphology ( Fig 17 ). Injection of ibotenic acid with subsequent infusion of vehicle resulted in a substantial (43%) loss of ChAT-immunoreactive neurons ( Fig 18 , bar 4) over an approximately 1 mm radius from the injection site in the left basal nucleus.
  • the sham-operated control group receiving vehicle infusion showed an increase in the percentage of ChAT-positive cell bodies in the left basal nucleus relative to the right basal nucleus ( Fig 18 , bar 1).
  • Ibotenate lesioned animals that received GLP-1 or exendin-4 infusions resulted in a decreased loss of ChAT-immunoreactive cell bodies in the left basal nucleus relative to those lesioned animals that received vehicle infusion. More specifically, infusion of exendin-4 produced a decrease in the loss of ChAT-immunoreactive cell bodies in the left basal nucleus, from 43% as was apparent following vehicle infusion, to just 24% below that of the right basal nucleus ( Fig 18 , bar 5).
  • GLP-1 infusion resulted in more striking reversal effects, decreasing the loss of ChAT-positive immunoreactive cell bodies in the left basal nucleus to just 6% below that of the right basal nucleus ( Fig 18 , bar 6).
  • Multiple comparisons of peptide vs . vehicle treatment revealed significant improvements in ChAT-immunoreactivity in the left basal nucleus following infusion of exendin-4 (p ⁇ 0.05) and GLP-1 (p ⁇ 0.01) after an ibotenic acid lesion.
  • ChAT-positive immunoreactive neuronal counts for the sham vehicle group revealed a significant difference between left (586 ⁇ 32) and right (478 ⁇ 40) basal nuclei ChAT-positive cell counts (p ⁇ 0.01).
  • Pressure effects from cannula implantation and treatment delivery may account for the apparent decrease in ChAT-immunoreactivity in the right basal nucleus.
  • Glucagon-like peptide-1 decreases amyloid- ⁇ peptide (A ⁇ ) production and protects neurons against death induced by A ⁇ and iron
  • PC12 cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated horse serum, 5% heat-inactivated fetal bovine serum (FBS), and 25mM
  • Hepes buffer as described elsewhere (Lahiri et. al., 2000). All culture media and sera were obtained from MediaTech Inc. (Herndon, VA). Cells were seeded at approximately 2.0 x 10 6 cells / 60-mm dish, on cultureware coated in rat-tail collagen (Roche Molecular Biochemicals, Indianapolis, IN). Treatments in triplicate began 24 h after seeding, once cells were well attached. The medium was aspirated, and 3 ml of fresh low serum media containing 0.5% FBS with the appropriate compound(s) was added.
  • LDH lactate dehydrogenase
  • conditioned media and cell lysates prepared as described above
  • untreated low serum medium alone
  • treated PC12 cells were subjected to Western immunoblot analysis using the monoclonal antibody, 22C11 (Roche Molecular Biochemicals, Indianapolis, IN).
  • the antibody raised against E. Coli-made ⁇ APP whose epitope region has been assigned to residues 66-81 in the ectoplasmic cysteine-containing domain, recognizes all mature forms of ⁇ APP found in cell membranes, as well as carboxyl-truncated soluble forms secreted into conditioned media and ⁇ APP-like proteins (APLP).
  • Western immunoblots of cell lysates from treated or untreated cells revealed multiple higher molecular weight protein bands (100-140 kDa) that likely represent different isoforms of mature ⁇ APP ( ⁇ APP695 - ⁇ APP770) and/or their post-translationally modified derivatives.
  • Secreted APP was detected in conditioned media from treated and untreated cells, as a 110-120 kDa protein band, likely representing derivatives of ⁇ APP generated by either ⁇ - or ⁇ -secretase.
  • GLP-1 and analogues significantly decreased intracellular levels of ⁇ APP ( Fig 19B ; 10 ng/ml Ex4, bar 5 (p ⁇ 0.01), 1.0 ⁇ g/ml Ex4-WOT, bar 10 (p ⁇ 0.01), 3.3 ⁇ g/ml GLP-1, bar 11 (p ⁇ 0.01), 33 ⁇ g/ml GLP-1, bar 12 (p ⁇ 0.05) and 330 ⁇ g/ml GLP-1, bar 13 (p ⁇ 0.01).
  • the combination treatment of 5 ng/ml NGF and 0.1 ⁇ g/ml Ex4 significantly increased intracellular ⁇ APP levels relative to untreated cells ( Fig 19B , bar 6; p ⁇ 0.01).
  • conditioned media samples were assayed for A ⁇ 1-40.
  • the predominant A ⁇ peptide secreted is A ⁇ 1-40 and in similar studies using neuroblastoma cells (Lahiri et. al., 1998) A ⁇ 1-40 levels were reflective of effects on A ⁇ 1-42, making it unnecessary to look for A ⁇ 1-42 specific changes.
  • Tc 69.91 and 83.13 revealed significant increases in A ⁇ 1-40 levels following treatment with NGF alone (both p ⁇ 0.01) or in combination with exendin-4 (p ⁇ 0.01). Treatment with GLP-1, exendin-4 or exendin-4 WOT alone did not significantly alter levels of A ⁇ 1-40 production from that of the control cells.
  • Hippocampi were removed from embryonic day 18 Sprague-Dawley rats, and cells were dissociated by mild trypsination and trituration and seeded onto polyethyleneimine-coated plastic 35 mm diameter dishes at a density of approximately 150 cells/mm 2 of culture surface. Cultures were maintained in Neurobasal medium containing B-27 supplements (Gibco BRL, Carlsbad, CA), 2 mM L-glutamine, 1 mM Hepes and 0.001% gentamicin sulfate (Sigma, St. Louis, MO) in a 6% CO 2 /94% room air atmosphere at 37°C.
  • B-27 supplements Gibco BRL, Carlsbad, CA
  • 2 mM L-glutamine 1 mM Hepes
  • gentamicin sulfate Sigma, St. Louis, MO
  • the hippocampal cultures When maintained under these conditions, the hippocampal cultures consisted of approximately 95% neurons and 5% astrocytes as determined by immunostaining with antibodies against the neuronal antigen NeuN and the astrocyte protein glial fibrillary acidic protein.
  • a ⁇ 1-42 was purchased from Bachem (Torrance, CA) and was prepared as a 1 mM stock solution in sterile water.
  • FeSO 4 was prepared as a 200 ⁇ M stock in sterile water.
  • GLP-1 and exendin-4 were prepared as 500X stocks in saline.
  • Neuronal survival was quantified as described previously (Mark et. al., 1997). Briefly, viable neurons in premarked fields (10X objective) were counted before experimental treatment and at specified time points thereafter. Hippocampal neurons were pretreated with GLP-1 (0, 1, 5, 10 and 20 nM) or exendin-4 (0, 50, 100, 200 and 500 nM) for 2 hours. Cultures were then exposed to 2 ⁇ M A ⁇ (1-42) or 1 ⁇ M Fe 2+ for 24 hours. Neurons with intact neurites of uniform diameter and a cell body with a smooth round appearance were considered viable, whereas neurons with fragmented neurites and vacuolated soma were considered non-viable. The counting was done without knowledge of culture treatment history.
  • GLP-1 or exendin-4 could protect neurons against cell death induced by A ⁇ 1-42 and/or Fe 2+ .
  • cells were pretreated with increasing concentrations of GLP-1 and exendin-4, and then exposed to A ⁇ 1-42 or Fe 2+ .
  • GLP-1 protected neurons against death induced by A ⁇ 1-42 and Fe 2+ with a maximum effect occurring with 10 nM GLP-1.
  • Exendin-4 also protected neurons against death induced by A ⁇ 1-42 and Fe 2+ , but was less potent, with a maximum effect occurring with 200 nM exendin-4.
  • a purified polypeptide the amino acid sequence of which comprises SEQ ID NO:5.
  • a method of treating a subject with diabetes comprising administering to the subject the polypeptide as defined above in an amount that has an insulinotropic effect.
  • a purified polypeptide the amino acid sequence of which consists of SEQ ID NO:5.
  • a purified polypeptide the amino acid sequence of which comprises SEQ ID NO:6.
  • polypeptide as defined above, wherein the polypeptide is insulinotropic.
  • a method of treating a subject with diabetes comprising administering to the subject the polypeptide of claim 6 in an amount that has an insulinotropic effect.
  • a purified polypeptide the amino acid sequence of which consists of SEQ ID NO:6.
  • a purified polypeptide the amino acid sequence of which consists of SEQ ID NO:7.
  • polypeptide as defined above, wherein the polypeptide is insulinotropic.
  • a method of treating a subject with diabetes comprising administering to the subject the polypeptide as defined above in an amount that has an insulinotropic effect.
  • polypeptide comprises the amino acid sequence of GLP-1, a GLP-1 analogue, exendin-4, or an exendin-4 analogue with a spacer between the amino acid residues comparable to residues 7 and 8 or residues 8 and 9 of GLP-1.
  • polypeptide as defined above, wherein the polypeptide is insulinotropic.
  • the polypeptide as defined above, wherein the spacer is a 6-aminohexanoic acid spacer.
  • the polypeptide as defined above, wherein the 6-aminohexanoic acid spacer comprises less than four 6-aminohexanoic acid residues.
  • polypeptide as defined above, the amino acid sequence of which comprises SEQ ID NO:8.
  • polypeptide as defined above, the amino acid sequence of which comprises SEQ ID NO:42.
  • polypeptide as defined above, the amino acid sequence of which comprises SEQ ID NO:43.
  • polypeptide as defined above, the amino acid sequence of which comprises SEQ ID NO:44.
  • polypeptide as defined above, the amino acid sequence of which comprises SEQ ID NO:45.
  • polypeptide as defined above, the amino acid sequence of which comprises SEQ ID NO:46.
  • polypeptide as defined above, the amino acid sequence of which comprises SEQ ID NO:47.
  • polypeptide as defined above, the amino acid sequence of which comprises SEQ ID NO: 48.
  • polypeptide as defined above, wherein the amino acid sequence of the GLP-1, GLP-1 analogue, exendin-4, or exendin-4 analogue comprises a sequence selected from the group consisting of SEQ ID NO:1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 25, 33, 42, 43, 44, 45, 46, 47, and 48.
  • a method of treating a subject with diabetes comprising administering to the subject the polypeptide of claim 15 in an amount that has an insulinotropic effect.
  • a purified polypeptide the amino acid sequence of which comprises SEQ ID NO:9.
  • a method of treating a subject with diabetes comprising administering to the subject the polypeptide as defined above in an amount that has an insulinotropic effect.
  • a purified polypeptide the amino acid sequence of which consists of SEQ ID NO:9.
  • a purified polypeptide the amino acid sequence of which comprises SEQ ID NO:10.
  • a method of treating a subject with diabetes comprising administering to the subject the polypeptide as defined above in an amount that has an insulinotropic effect.
  • a purified polypeptide the amino acid sequence of which consists of SEQ ID NO:10.
  • a purified polypeptide the amino acid sequence of which comprises SEQ ID NO:11.
  • a method of treating a subject with diabetes comprising administering to the subject the polypeptide as defiend above in an amount that has an insulinotropic effect.
  • a method of treating a subject with diabetes comprising administering to the subject the polypeptide as defined above in an amount that has an insulinotropic effect.
  • a purified polypeptide the amino acid sequence of which consists of SEQ ID NO:13.
  • a method of treating a subject with diabetes comprising administering to the subject the polypeptide as defined above in an amount that has an insulinotropic effect.
  • a purified polypeptide the amino acid sequence of which consists of SEQ ID NO:14.
  • a method of treating a subject with diabetes comprising administering to the subject the polypeptide as defined above in an amount that has an insulinotropic effect.
  • a purified polypeptide the amino acid sequence of which consists of SEQ ID NO:15.
  • polypeptide as defined above, wherein the polypeptide is insulinotropic.
  • a method of treating a subject with diabetes comprising administering to the subject the polypeptide as defined above in an amount that has an insulinotropic effect.
  • a purified polypeptide the amino acid sequence of which comprises SEQ ID NO:25.
  • polypeptide as defined above, wherein the polypeptide is insulinotropic.
  • a method of treating a subject with diabetes comprising administering to the subject the polypeptide as defined above in an amount that has an insulinotropic effect.
  • a purified polypeptide the amino acid sequence of which consists of SEQ ID NO:25.
  • a purified polypeptide the amino acid sequence of which comprises SEQ ID NO:33.
  • polypeptide as defined above, wherein the polypeptide is insulinotropic.
  • a method of treating a subject with diabetes comprising administering to the subject the polypeptide as defined above in an amount that has an insulinotropic effect.
  • a purified polypeptide the amino acid sequence of which consists of SEQ ID NO:33.
  • a method of reducing neuronal death comprising contacting one or more neurons with a polypeptide comprising GLP-1, exendin-4, or a neuroprotective GLP-1 or exendin-4 analogue.
  • polypeptide is selected from the group consisting of SEQ ID NO:1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 25, 33, 42, 43, 44, 45, 46, 47, and 48.
  • a method of promoting neuronal differentiation or proliferation comprising contacting one or more neurons or neuronal precursor cells with a polypeptide comprising GLP-1, exendin-4, or a differentiation-inducing or proliferation-inducing GLP-1 or exendin-4 analogue.
  • polypeptide is selected from the group consisting of SEQ ID NO:1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 25, 33, 42, 43, 44, 45, 46, 47, and 48.
  • a method of reducing formation or accumulation of amyloid, protein comprising contacting one or more neurons with a polypeptide comprising GLP-1, exendin-4, or a GLP-1 or exendin-4 analogue that affects -amyloid precursor protein metabolism.
  • polypeptide is selected from the group consisting of SEQ ID NO:1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 25, 33, 42, 43, 44, 45, 46, 47, and 48.
  • a method of promoting growth of neuronal processes comprising contacting one or more neurons with a polypeptide comprising GLP-1, exendin-4, or a process-promoting GLP-1 or exendin-4 analogue.
  • polypeptide is selected from the group consisting of SEQ ID NO:1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,25,33, 42, 43, 44, 45, 46, 47, and 48.
  • a method of treating a subject with a neurodegenerative condition or of reducing one or more symptoms of a neurodegenerative condition in a subj ect comprising administering to the subject a therapeutically effective amount of a polypeptide comprising GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analogue.
  • the neurodegenerative condition is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, stroke, multiple sclerosis, brain injury, spinal cord injury, and peripheral neuropathy.
  • a method of treating a subject with a neurotoxic injury or of reducing one or more symptoms of a neurotoxic injury in a subject comprising administering to the subject a therapeutically effective amount of a polypeptide comprising GLP-1, exendin-4, or a therapeutically effective GLP-1 or exendin-4 analogue.

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Families Citing this family (137)

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Publication number Priority date Publication date Assignee Title
US20050272652A1 (en) 1999-03-29 2005-12-08 Gault Victor A Peptide analogues of GIP for treatment of diabetes, insulin resistance and obesity
DK1294757T3 (da) * 2000-06-16 2007-03-19 Lilly Co Eli Glucagonlignende peptid 1-analoger
DE60228972D1 (de) 2001-07-31 2008-10-30 Us Gov Health & Human Serv Glp 1 exendin 4 peptidanaloga und deren verwendungen
EP2277910A1 (fr) 2001-12-21 2011-01-26 Human Genome Sciences, Inc. Protéines de fusion d'albumine
JP4282485B2 (ja) 2002-01-08 2009-06-24 イーライ リリー アンド カンパニー 伸長グルカゴン様ペプチド−１アナログ
GB0205693D0 (en) * 2002-03-09 2002-04-24 Astrazeneca Ab Chemical compounds
US7731947B2 (en) 2003-11-17 2010-06-08 Intarcia Therapeutics, Inc. Composition and dosage form comprising an interferon particle formulation and suspending vehicle
AU2004231461B2 (en) * 2003-03-19 2009-11-12 Eli Lilly And Company Polyethelene glycol linked GLP-1 compounds
EP1633384B1 (fr) * 2003-05-15 2012-03-14 Trustees Of Tufts College Analogues stables de glp-1
US7329725B1 (en) * 2003-10-29 2008-02-12 Nastech Pharmaceutical Company Inc. Phage displayed Trp cage ligands
EP1709071A4 (fr) * 2004-01-08 2007-05-30 Theratechnologies Inc Analogues peptidiques 1 semblables au glucagon a action de longue duree
EP1789440A4 (fr) 2004-02-11 2008-03-12 Amylin Pharmaceuticals Inc Motifs de la famille de polypeptides pancreatiques et polypeptides les renfermant
US8076288B2 (en) 2004-02-11 2011-12-13 Amylin Pharmaceuticals, Inc. Hybrid polypeptides having glucose lowering activity
US7893017B2 (en) 2004-10-07 2011-02-22 Novo Nordisk A/S Protracted GLP-1 compounds
JP5107713B2 (ja) 2004-10-07 2012-12-26 ノヴォ ノルディスク アー／エス 遅延性のエキセンディン−４化合物
EP1838336A2 (fr) * 2004-12-24 2007-10-03 Amylin Pharmaceuticals, Inc. Utilisation de glp-1 et d'agonistes de ce compose pour prevenir l'apoptose de myocytes cardiaques
US11246913B2 (en) 2005-02-03 2022-02-15 Intarcia Therapeutics, Inc. Suspension formulation comprising an insulinotropic peptide
WO2006083761A2 (fr) 2005-02-03 2006-08-10 Alza Corporation Solutions de solvant/polymere utilisees comme vehicules de suspension
US8263545B2 (en) 2005-02-11 2012-09-11 Amylin Pharmaceuticals, Inc. GIP analog and hybrid polypeptides with selectable properties
JP2008530130A (ja) 2005-02-11 2008-08-07 アミリン・ファーマシューティカルズ，インコーポレイテッド Ｇｉｐ類似体および選択可能な特性を備えるハイブリッドポリペプチド
EP1891106A2 (fr) * 2005-05-05 2008-02-27 Cadila Healthcare Ltd. Nouveaux composes utilises en tant qu'agonistes de glp-i
CN100429227C (zh) 2005-06-29 2008-10-29 常州制药厂有限公司 Exendin4多肽片段
EA014647B1 (ru) * 2005-08-11 2010-12-30 Амилин Фармасьютикалз, Инк. Гибридные полипептиды с селектируемыми свойствами
CA2617649A1 (fr) * 2005-08-11 2007-02-22 Amylin Pharmaceuticals, Inc. Polypeptides hybrides presentant des proprietes selectionnables
EP2497781A1 (fr) * 2005-09-08 2012-09-12 Trustees Of Tufts College Analogues de GLP-1 stabilisés
AU2006289259A1 (en) * 2005-09-08 2007-03-15 Uutech Limited Analogs of gastric inhibitory polypeptide as a treatment for age related decreased pancreatic beta cell function
EP1943274A2 (fr) * 2005-09-08 2008-07-16 Uutech Limited Traitement de l'obesite associee aux diabetes
JP5027137B2 (ja) 2005-09-14 2012-09-19 武田薬品工業株式会社 ジペプチジルペプチダーゼ阻害剤の投与
KR101368988B1 (ko) 2005-09-16 2014-02-28 다케다 야쿠힌 고교 가부시키가이샤 디펩티딜 펩티다제 억제제
DK1767545T3 (da) 2005-09-22 2010-03-15 Biocompatibles Uk Ltd GLP-1 (Glucagon-lignende peptid-1)-fusionspolypeptider med forøget peptidaseresistens
CA2628241C (fr) 2005-11-07 2016-02-02 Indiana University Research And Technology Corporation Analogues de glucagon a solubilite et a stabilite physiologiques ameliorees
WO2007112347A1 (fr) 2006-03-28 2007-10-04 Takeda Pharmaceutical Company Limited Inhibiteurs de la dipeptidyl peptidase
JP2009532422A (ja) * 2006-04-03 2009-09-10 ノボ・ノルデイスク・エー／エス Ｇｌｐ−１ペプチドアゴニスト
EP2574624A1 (fr) * 2006-04-20 2013-04-03 Amgen Inc. Composés GLP-1
EP1854455B1 (fr) 2006-05-10 2009-10-07 Biocompatibles UK Limited Microcapsules sphériques contenant des peptides du type GLP-1, leur production et leur utilisation
KR101106510B1 (ko) 2006-05-30 2012-01-20 인타르시아 세라퓨틱스 인코포레이티드 투피스, 내부채널 삼투압 전달 시스템 유동 조절기
MX2009001114A (es) 2006-08-09 2009-02-10 Intarcia Therapeutics Inc Sistemas de suministro osmotico y ensambles de piston.
US8497240B2 (en) 2006-08-17 2013-07-30 Amylin Pharmaceuticals, Llc DPP-IV resistant GIP hybrid polypeptides with selectable properties
US8324383B2 (en) 2006-09-13 2012-12-04 Takeda Pharmaceutical Company Limited Methods of making polymorphs of benzoate salt of 2-[[6-[(3R)-3-amino-1-piperidinyl]-3,4-dihydro-3-methyl-2,4-dioxo-1(2H)-pyrimidinyl]methyl]-benzonitrile
TW200838536A (en) 2006-11-29 2008-10-01 Takeda Pharmaceutical Polymorphs of succinate salt of 2-[6-(3-amino-piperidin-1-yl)-3-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethy]-4-fluor-benzonitrile and methods of use therefor
AU2008205229B2 (en) 2007-01-05 2014-03-27 Indiana University Research And Technology Corporation Glucagon analogs exhibiting enhanced solubility in physiological pH buffers
RU2413528C2 (ru) 2007-01-18 2011-03-10 Открытое Акционерное Общество "Валента Фармацевтика" Лекарственный препарат для лечения сахарного диабета на основе экзенатида и даларгина, применение и способ лечения
AU2008216265B2 (en) 2007-02-15 2014-04-03 Indiana University Research And Technology Corporation Glucagon/GLP-1 receptor co-agonists
US8093236B2 (en) 2007-03-13 2012-01-10 Takeda Pharmaceuticals Company Limited Weekly administration of dipeptidyl peptidase inhibitors
EP1972349A1 (fr) * 2007-03-21 2008-09-24 Biocompatibles UK Limited Peptides à fusion GLP-1 associée à un/plusieurs polymères, leur production et utilisation
EP1975176A1 (fr) * 2007-03-27 2008-10-01 Biocompatibles UK Limited Nouveaux peptides à fusion GLP-1, leur production et utilisation
RU2440097C2 (ru) 2007-04-23 2012-01-20 Интарсия Терапьютикс, Инк. Способ лечения диабета ii типа и ожирения, осмотическое устройство для доставки и способ его изготовления
EP2190872B1 (fr) 2007-09-05 2018-03-14 Novo Nordisk A/S Dérivés de glucagon-like peptide-1 et leur utilisation pharmaceutique
WO2009030771A1 (fr) * 2007-09-05 2009-03-12 Novo Nordisk A/S Peptides dérivés avec a-b-c-d et utilisation thérapeutique de ceux-ci
CN101842386A (zh) 2007-09-05 2010-09-22 诺沃-诺迪斯克有限公司 截短的glp-1衍生物和它们的治疗用途
GB0717399D0 (en) 2007-09-07 2007-10-17 Uutech Ltd Use of GLP-1 analogues for the treatment of disorders associated with dysfunctional synaptic transmission
GB0717388D0 (en) * 2007-09-07 2007-10-17 Uutech Ltd Use of GIP for the treatment of disorders associated with dysfunctional synaptic transmission
MX2010004297A (es) 2007-10-30 2010-05-03 Univ Indiana Res & Tech Corp Antagonistas de glucagon.
CA2702289A1 (fr) 2007-10-30 2009-05-07 Indiana University Research And Technology Corporation Composes presentant une activite d'antagoniste de glucagon et d'agoniste du glp-1
EP2240155B1 (fr) 2008-02-13 2012-06-06 Intarcia Therapeutics, Inc Dispositifs, formulations et méthodes d administration de plusieurs agents bénéfiques
CA2726903A1 (fr) * 2008-06-03 2009-12-10 Trustees Of Tufts College Derives du glp-1 a action prolongee et procedes de traitement d'un dysfonctionnement cardiaque
NZ589847A (en) 2008-06-17 2013-01-25 Univ Indiana Res & Tech Corp Glucagon/glp-1 receptor co-agonists
US8450270B2 (en) 2008-06-17 2013-05-28 Indiana University Research And Technology Corporation Glucagon analogs exhibiting enhanced solubility and stability in physiological pH buffers
CN104945500B (zh) 2008-06-17 2019-07-09 印第安纳大学研究及科技有限公司 基于gip的混合激动剂用于治疗代谢紊乱和肥胖症
RS59913B1 (sr) 2008-10-17 2020-03-31 Sanofi Aventis Deutschland Kombinacija insulina i glp-1-agonista
JP2012512903A (ja) 2008-12-19 2012-06-07 インディアナ ユニバーシティー リサーチ アンド テクノロジー コーポレーション アミド系グルカゴンスーパーファミリーペプチドプロドラッグ
BRPI0923853A2 (pt) * 2008-12-29 2016-01-12 Panacea Biotec Ltd análogos de gpl-1 e usos dos mesmos.
EP2413955A4 (fr) * 2009-04-01 2012-12-26 Amylin Pharmaceuticals Inc Composés agonistes des récepteurs au glp-1 présentant des contraintes conformationnelles au niveau de leur extrémité n-terminale
WO2010118034A2 (fr) * 2009-04-06 2010-10-14 Board Of Regents, The University Of Texas System Analogues peptidiques cycliques utilisés en imagerie non invasive des cellules bêta du pancréas
RU2012101274A (ru) 2009-06-16 2013-07-27 Индиана Юниверсити Рисерч Энд Текнолоджи Корпорейшн Соединения глюкагона, активные в отношении рецептора gip
US20120148586A1 (en) 2009-08-27 2012-06-14 Joyce Ching Tsu Chou Glucagon-like protein-1 receptor (glp-1r) agonists for treating autoimmune disorders
SI2462246T1 (en) 2009-09-28 2018-01-31 Intarcia Therapeutics, Inc. Fast-setting and / or cessation of substantially unchanged delivery of the product
KR101972301B1 (ko) 2009-11-13 2019-04-25 사노피-아벤티스 도이칠란트 게엠베하 Ｇｌｐ－１ 작용제, 인슐린 및 메티오닌을 포함하는 약제학적 조성물
BR112012011403B8 (pt) 2009-11-13 2021-05-25 Sanofi Aventis Deutschland composição farmacêutica líquida compreendendo um agonista glp-1 e metionina e uso da mesma
WO2011075393A2 (fr) 2009-12-18 2011-06-23 Indiana University Research And Technology Corporation Co-agonistes du récepteur du glucagon/glp-i
WO2011094337A1 (fr) 2010-01-27 2011-08-04 Indiana University Research And Technology Corporation Conjugués d'antagoniste du glucagon et d'agoniste du gip et compositions pour le traitement de troubles métaboliques et de l'obésité
EP2569000B1 (fr) 2010-05-13 2017-09-27 Indiana University Research and Technology Corporation Peptides de la superfamille des glucagons présentant une activité de récepteur nucléaire d'hormone
CN103179976A (zh) 2010-05-13 2013-06-26 印第安纳大学研究及科技有限公司 呈现g蛋白偶联受体活性的胰高血糖素超家族肽
KR20130102470A (ko) 2010-06-24 2013-09-17 인디애나 유니버시티 리서치 앤드 테크놀로지 코퍼레이션 아미드계 글루카곤 슈퍼패밀리 펩티드 프로드러그
CA2809321C (fr) 2010-08-30 2018-08-14 Sanofi-Aventis Deutschland Gmbh Utilisation de lixisenatide pour fabrication de medicament pour traitement du diabete sucre de type 2
US9023986B2 (en) 2010-10-25 2015-05-05 Hoffmann-La Roche Inc. Glucose-dependent insulinotropic peptide analogs
AU2011343190B2 (en) 2010-12-16 2016-12-08 Novo Nordisk A/S Solid compositions comprising a GLP-1 agonist and a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid
US8507428B2 (en) 2010-12-22 2013-08-13 Indiana University Research And Technology Corporation Glucagon analogs exhibiting GIP receptor activity
US9050347B2 (en) 2011-01-25 2015-06-09 Viviabiotech, S.L. 1,2,4-oxadiazole derivatives as drugs modulating the GLP-1 peptide receptor
US20120208755A1 (en) 2011-02-16 2012-08-16 Intarcia Therapeutics, Inc. Compositions, Devices and Methods of Use Thereof for the Treatment of Cancers
CN106117343B (zh) 2011-04-12 2020-11-03 诺沃—诺迪斯克有限公司 双酰化glp-1衍生物
CA2833623A1 (fr) 2011-04-20 2012-10-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Transfert du gene exendine 4 medie par aav vers les glandes salivaires pour proteger des sujets contre le diabete ou l'obesite
US9821032B2 (en) 2011-05-13 2017-11-21 Sanofi-Aventis Deutschland Gmbh Pharmaceutical combination for improving glycemic control as add-on therapy to basal insulin
PE20140724A1 (es) 2011-06-22 2014-07-10 Univ Indiana Res & Tech Corp Coagonistas del receptor de glucagon/glp-1
US9309301B2 (en) 2011-06-22 2016-04-12 Indiana University Research And Technology Corporation Glucagon/GLP-1 receptor co-agonists
HUE027989T2 (en) 2011-08-29 2016-11-28 Sanofi Aventis Deutschland A pharmaceutical composition for use in glycemic control in patients with type 2 diabetes
AR087744A1 (es) * 2011-09-01 2014-04-16 Sanofi Aventis Deutschland Composicion farmaceutica para uso en el tratamiento de una enfermedad neurodegenerativa
KR20140097151A (ko) 2011-11-17 2014-08-06 인디애나 유니버시티 리서치 앤드 테크놀로지 코퍼레이션 글루코코르티코이드 수용체 활성을 나타내는 글루카곤 슈퍼패밀리 펩티드
US9388212B2 (en) 2013-02-21 2016-07-12 Chemical & Biopharmaceutical Laboratories Of Patras S.A. Solid phase peptide synthesis via side chain attachment
DK2827845T3 (en) 2012-03-22 2019-04-01 Novo Nordisk As COMPOSITIONS INCLUDING A PROCEDURE AND PREPARING THEREOF
US10933120B2 (en) 2012-03-22 2021-03-02 Novo Nordisk A/S Compositions of GLP-1 peptides and preparation thereof
US9993430B2 (en) 2012-06-20 2018-06-12 Novo Nordisk A/S Tablet formulation comprising semaglutide and a delivery agent
WO2013192129A1 (fr) 2012-06-21 2013-12-27 Indiana University Research And Technology Corporation Analogues du glucagon présentant une activité sur le récepteur du gip
UA116217C2 (uk) 2012-10-09 2018-02-26 Санофі Пептидна сполука як подвійний агоніст рецепторів glp1-1 та глюкагону
US20130303436A1 (en) * 2012-12-06 2013-11-14 Stealth Peptides Internatioanl, Inc. Peptide therapeutics and methods for using same
JP2016503046A (ja) * 2012-12-19 2016-02-01 ノヴォ ノルディスク アー／エス コレステロール流出活性を有する新規ｇｌｐ−１受容体アゴニスト
WO2014096150A1 (fr) 2012-12-21 2014-06-26 Sanofi Agonistes du glp1/gip double ou du glp1/gip/glucagon trigonal
JP6382488B2 (ja) * 2013-02-21 2018-08-29 ケミカル アンド バイオファーマシューティカル ラボラトリーズ オブ パトラ エス．エー． 側鎖結合を介する固相ペプチド合成
US10028902B2 (en) 2013-11-08 2018-07-24 Baylor Research Institute Nuclear localization of GLP-1 stimulates myocardial regeneration and reverses heart failure
US10626156B2 (en) 2013-12-06 2020-04-21 Jie Han Bioreversable promoieties for nitrogen-containing and hydroxyl-containing drugs
EP3080154B1 (fr) 2013-12-13 2018-02-07 Sanofi Agonistes doubles du récepteur glp-1/gip
EP3080152A1 (fr) 2013-12-13 2016-10-19 Sanofi Analogues peptidiques de l'exendine 4 non acylés
TW201609795A (zh) 2013-12-13 2016-03-16 賽諾菲公司 作為雙重ｇｌｐ－１／ｇｉｐ受體促效劑的艾塞那肽－４（ｅｘｅｎｄｉｎ－４）胜肽類似物
TW201609797A (zh) 2013-12-13 2016-03-16 賽諾菲公司 雙重ｇｌｐ－１／升糖素受體促效劑
TW201625668A (zh) 2014-04-07 2016-07-16 賽諾菲公司 作為胜肽性雙重ｇｌｐ－１／昇糖素受體激動劑之艾塞那肽－４衍生物
TW201625670A (zh) 2014-04-07 2016-07-16 賽諾菲公司 衍生自ｅｘｅｎｄｉｎ－４之雙重ｇｌｐ－１／升糖素受體促效劑
TW201625669A (zh) 2014-04-07 2016-07-16 賽諾菲公司 衍生自艾塞那肽－４（Ｅｘｅｎｄｉｎ－４）之肽類雙重ＧＬＰ－１／升糖素受體促效劑
US9932381B2 (en) 2014-06-18 2018-04-03 Sanofi Exendin-4 derivatives as selective glucagon receptor agonists
GB201415598D0 (en) 2014-09-03 2014-10-15 Univ Birmingham Elavated Itercranial Pressure Treatment
US9889085B1 (en) 2014-09-30 2018-02-13 Intarcia Therapeutics, Inc. Therapeutic methods for the treatment of diabetes and related conditions for patients with high baseline HbA1c
SI3229828T1 (sl) 2014-12-12 2023-06-30 Sanofi-Aventis Deutschland Gmbh Formulacija s fiksnim razmerjem inzulin glargin/liksisenatid
TW201639878A (zh) 2014-12-30 2016-11-16 韓美藥品股份有限公司 具有改善之安定性的升糖素衍生物
TWI748945B (zh) 2015-03-13 2021-12-11 德商賽諾菲阿凡提斯德意志有限公司 第２型糖尿病病患治療
TW201705975A (zh) 2015-03-18 2017-02-16 賽諾菲阿凡提斯德意志有限公司 第２型糖尿病病患之治療
US10993993B2 (en) 2015-05-28 2021-05-04 Immunoforge Co., Ltd. Pharmaceutical composition for treating muscle atrophy or sarcopenia including glucagon-like peptide (GLP-1) or GLP-1 receptor agonist
KR101661332B1 (ko) 2015-05-28 2016-09-29 (의료)길의료재단 글루카곤 유사 펩타이드-1 수용체 항진제를 포함하는 근감소증 치료용 약학 조성물
EP3302354B1 (fr) 2015-06-03 2023-10-04 i2o Therapeutics, Inc. Systèmes de mise en place d'implant
AR105319A1 (es) 2015-06-05 2017-09-27 Sanofi Sa Profármacos que comprenden un conjugado agonista dual de glp-1 / glucagón conector ácido hialurónico
JP6882210B2 (ja) 2015-06-30 2021-06-02 ハンミ ファーマシューティカル カンパニー リミテッド グルカゴン誘導体及びその持続型結合体を含む組成物
AR105284A1 (es) 2015-07-10 2017-09-20 Sanofi Sa Derivados de exendina-4 como agonistas peptídicos duales específicos de los receptores de glp-1 / glucagón
PL3393496T3 (pl) 2015-12-23 2024-04-22 The Johns Hopkins University Długo działający agonista glp-1r jako terapia stanów neurologicznych i neurodegeneracyjnych
HRP20220995T1 (hr) * 2015-12-31 2022-11-11 Hanmi Pharm. Co., Ltd. Trostruki aktivator koji aktivira glukagonski, glp-1 i gip receptore
EP3733694A1 (fr) 2016-05-16 2020-11-04 Intarcia Therapeutics, Inc Polypeptides sélectifs pour le récepteur du glucagon et méthodes pour leur utilisation
USD840030S1 (en) 2016-06-02 2019-02-05 Intarcia Therapeutics, Inc. Implant placement guide
USD860451S1 (en) 2016-06-02 2019-09-17 Intarcia Therapeutics, Inc. Implant removal tool
TWI757305B (zh) 2016-06-29 2022-03-11 南韓商韓美藥品股份有限公司 升糖素衍生物、其接合物、及包含其之組成物、及其醫療用途
CN110191717A (zh) 2016-10-20 2019-08-30 佩特通公司 向中枢神经系统递送神经保护性多肽的方法
MX2019008006A (es) 2017-01-03 2019-08-29 Intarcia Therapeutics Inc Metodos que comprenden la administracion continua de un agonista del receptor de glp-1 y la co-administracion de un farmaco.
TWI804571B (zh) 2018-02-02 2023-06-11 丹麥商諾佛 儂迪克股份有限公司 包含glp-1促效劑及n-(8-(2-羥苯甲醯基)胺基)辛酸之鹽的固體組成物
CN110452952B (zh) * 2018-05-08 2024-01-16 宜昌东阳光长江药业股份有限公司 一种glp-1类似物生物活性的检测方法
KR101921085B1 (ko) 2018-05-21 2018-11-22 이뮤노포지 주식회사 글루카곤 유사 펩타이드-1(glp-1), glp-1 유래 펩타이드, 또는 glp-1 분해 억제제를 포함하는 근감소증 또는 근위축증 치료용 약학 조성물
KR102119188B1 (ko) * 2018-11-13 2020-06-08 이뮤노포지 주식회사 글루카곤 유사 펩타이드-1(glp-1), glp-1 유래 펩타이드, 또는 glp-1 분해 억제제를 포함하는 근감소증 또는 근위축증 치료용 약학 조성물
US20220047667A1 (en) * 2019-02-28 2022-02-17 Nitta Getain Inc. Brain function regulating agent, and food or beverage product containing same
WO2020214012A1 (fr) * 2019-04-19 2020-10-22 한미약품 주식회사 Composition pharmaceutique préventive ou thérapeutique contre l'hyperlipidémie comprenant un triple agoniste agissant sur tous les récepteurs du glucagon, du glp-1 et du gip, ou conjugué de ceux-ci, et procédé préventif ou thérapeutique
WO2020214013A1 (fr) * 2019-04-19 2020-10-22 한미약품 주식회사 Utilisation thérapeutique, pour l'hyperlipidémie, d'un triple agoniste ayant une activité par rapport à tous les récepteurs du glucagon, glp -1 et gip, ou conjugué de ceux-ci
MX2022001975A (es) 2019-08-16 2022-03-11 Applied Molecular Transport Inc Composiciones, formulaciones y produccion y purificacion de interleucinas.
JP2023510864A (ja) * 2020-01-13 2023-03-15 ハンミ ファーマシューティカル カンパニー リミテッド グルカゴン、ｇｌｐ－１及びｇｉｐ受容体の全てに対して活性を有する三重活性体の持続型結合体の肺疾患の治療的使用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3710795A (en) 1970-09-29 1973-01-16 Alza Corp Drug-delivery device with stretched, rate-controlling membrane
WO2000066142A2 (fr) 1999-04-30 2000-11-09 Bionebraska, Inc. Intervention metabolique a l'aide de glp-1 ou de ses analogues biologiquement actifs pour ameliorer la fonction cerebrale apres ischemie et reperfusion
WO2000069911A1 (fr) * 1999-05-17 2000-11-23 Conjuchem, Inc. Peptides insulinotropes a longue duree d'action

Family Cites Families (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5424286A (en) * 1993-05-24 1995-06-13 Eng; John Exendin-3 and exendin-4 polypeptides, and pharmaceutical compositions comprising same
EP0658568A1 (fr) 1993-12-09 1995-06-21 Eli Lilly And Company Peptides insulinotropes de type glucagon, compositions les contenant et méthode de préparation
GB9409496D0 (en) 1994-05-12 1994-06-29 London Health Ass Method for improving glycaemic control in diabetes
US5574008A (en) * 1994-08-30 1996-11-12 Eli Lilly And Company Biologically active fragments of glucagon-like insulinotropic peptide
DK0915910T3 (da) * 1996-06-05 2006-05-22 Roche Diagnostics Gmbh Exendin-analoger, fremgangsmåder til fremstilling deraf samt lægemidlter der indeholder disse
CA2277112C (fr) 1997-01-07 2008-08-26 Amylin Pharmaceuticals, Inc. Utilisation d'exendines et de leurs agonistes pour reduire la consommation alimentaire
US6268479B1 (en) * 1997-03-12 2001-07-31 The Trustees Of Columbia University In The City Of New York Intracellular amyloid-beta peptide binding (ERAB) polypeptide
EP2823812A1 (fr) 1998-02-02 2015-01-14 Trustees Of Tufts College Inhibiteurs de dipeptidylpeptidase-IV pour leur utilisation dans le traitement du diabète de type II
JP4677095B2 (ja) 1998-02-13 2011-04-27 アミリン・ファーマシューティカルズ，インコーポレイテッド エキセンジンおよびｇｌｐ−１の変力および利尿効果
EP1056774A1 (fr) 1998-02-27 2000-12-06 Novo Nordisk A/S Derives de glp-1 tronques a l'extremite n-terminale
EP1062229A1 (fr) 1998-03-09 2000-12-27 Zealand Pharmaceuticals A/S Conjugues peptidiques pharmacologiquement actifs ayant une tendance reduite a l'hydrolyse enzymatique
DE19828113A1 (de) 1998-06-24 2000-01-05 Probiodrug Ges Fuer Arzneim Prodrugs von Inhibitoren der Dipeptidyl Peptidase IV
JP4624558B2 (ja) 1998-08-10 2011-02-02 ザ ガバメント オブ ザ ユナイテッド ステイツ オブ アメリカ， アズ レプレゼンテッド バイ ザ セクレタリー， デパートメント オブ ヘルス アンド ヒューマン サービシーズ ＧＬＰ−１またはＥｘｅｎｄｉｎ−４による、非インスリン産生細胞のインスリン産生細胞への分化、およびその使用
MY155270A (en) 1998-09-24 2015-09-30 Lilly Co Eli Use of glp-1 or analogs in treatment of stroke
KR100452417B1 (ko) * 1998-12-07 2004-10-12 소시에떼 더 콘세이유 더 레세르세 에 다플리까띠옹 시엔띠피끄, 에스.아.에스. Ｇｌｐ-1의 유사체
PL208751B1 (pl) * 1998-12-07 2011-06-30 Sod Conseils Rech Applic Związki stanowiące analogi GLP-1, kompozycje zawierające takie związki oraz zastosowanie takich związków
JP3136356B2 (ja) 1998-12-15 2001-02-19 農林水産省家畜衛生試験場長 改変型シグナルペプチドをコードするｄｎａ
CN100356978C (zh) 1999-01-14 2007-12-26 安米林药品公司 新型exendin激动剂制剂及其给药方法
EP1189629B1 (fr) 1999-02-10 2010-09-29 Curis, Inc. Peptide yy (pyy) pour traiter des troubles metaboliques du glucose
US6506724B1 (en) 1999-06-01 2003-01-14 Amylin Pharmaceuticals, Inc. Use of exendins and agonists thereof for the treatment of gestational diabetes mellitus
EP1076066A1 (fr) * 1999-07-12 2001-02-14 Zealand Pharmaceuticals A/S Peptides abaissant le taux de glucose sanguin
KR20020060227A (ko) * 1999-11-10 2002-07-16 다케다 야쿠힌 고교 가부시키가이샤 체중 증가 억제제
US6278018B1 (en) 1999-12-14 2001-08-21 Surmodics, Inc. Surface coating agents
ES2227115T5 (es) * 2000-01-10 2014-10-30 Amylin Pharmaceuticals, Inc. Utilización de exendinas y sus agonistas para el tratamiento de la hipertrigliceridemia
AU2001232337A1 (en) * 2000-02-18 2001-08-27 Kanagawa Academy Of Science And Technology Pharmaceutical composition, reagent and method for intracerebral delivery of pharmaceutically active ingredient or labeling substance
EP1136071A3 (fr) * 2000-03-22 2003-03-26 Pfizer Products Inc. Utilisation d'inhibiteurs de la glycogène phosphorylase
US20020115605A1 (en) * 2000-08-24 2002-08-22 Matthew During Novel peptide with effects on cerebral health
DK1724284T3 (da) 2000-12-07 2009-11-02 Lilly Co Eli GLP-1 fusionsproteiner
DE60228972D1 (de) 2001-07-31 2008-10-30 Us Gov Health & Human Serv Glp 1 exendin 4 peptidanaloga und deren verwendungen
EP1532261B1 (fr) * 2002-05-24 2010-02-10 Medtronic, Inc. Procedes et constructions d'adn pour la production a haut rendement de polypeptides
US6969702B2 (en) * 2002-11-20 2005-11-29 Neuronova Ab Compounds and methods for increasing neurogenesis
DOP2006000008A (es) * 2005-01-10 2006-08-31 Arena Pharm Inc Terapia combinada para el tratamiento de la diabetes y afecciones relacionadas y para el tratamiento de afecciones que mejoran mediante un incremento de la concentración sanguínea de glp-1
CN102847141A (zh) 2005-09-28 2013-01-02 文特里亚生物科学公司 用于肠道病症和/或补水的口服制剂
JP2013007734A (ja) 2011-01-17 2013-01-10 Nippon Dempa Kogyo Co Ltd 外力検出装置及び外力検出センサー

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3710795A (en) 1970-09-29 1973-01-16 Alza Corp Drug-delivery device with stretched, rate-controlling membrane
WO2000066142A2 (fr) 1999-04-30 2000-11-09 Bionebraska, Inc. Intervention metabolique a l'aide de glp-1 ou de ses analogues biologiquement actifs pour ameliorer la fonction cerebrale apres ischemie et reperfusion
WO2000069911A1 (fr) * 1999-05-17 2000-11-23 Conjuchem, Inc. Peptides insulinotropes a longue duree d'action

Non-Patent Citations (62)

* Cited by examiner, † Cited by third party
Title
"Biology 3: Protection of Functional Groups in Peptide Synthesis", 1981, ACADEMIC PRESS, article "The Peptides: Analysis, Synthesis"
"Physician's Guide to Insulin Dependent [Type I] Diabetes Mellitus: Diagnosis and Treatment", 1988, AMERICAN DIABETES ASSOCIATION
"Remington's Pharmaceutical Sciences", MACK PUBLISHING CO.
"The Peptides: Analysis, Synthesis, Biology, Vol. 3: Protection of Functional Groups in Peptide Synthesis", vol. 3, 1981, ACADEMIC PRESS
ABERCROMBIE M: "Estimation of nuclear population from microtome sections", ANAT REC, vol. 94, 1946, pages 239 - 247
BRESSLER ET AL.: "Pharmacological regulation of blood glucose levels in non-insulin dependent diabetes", ARCH. INT. MED., vol. 157, 1997, pages 836 - 848
CALVO ET AL.: "Structural characterization by affinity cross-linking of glucagon-like peptide-1 (7-36) amide receptor in rat brain", J. NEUROCHEM., vol. 64, no. 1, 1995, pages 299 - 306
CAMPOS ET AL.: "Divergent tissue-specific and developmental expression of receptors for glucagon and glucagon-like peptide-1 in the mouse", ENDOCRINOLOGY, vol. 134, 1994, pages 2156 - 64
CHEN ET AL.: "Tissue-specific expression of unique mRNAs that encode pro-glucagon-derived peptides or exendin-4 in the lizard", J. BIOL. CHEM., vol. 272, 1997, pages 4108 - 4115
DE ORE ET AL.: "The effect of GLP-1 on insulin release in young and old rats in the fasting state and during an intravenous glucose tolerance test", J. GERONTOL., vol. 52, 1997, pages B245 - 249
DRUCKER D J: "Minireview: The glucagon-like peptides", ENDOCRINOLOGY, BALTIMORE, MD, US, vol. 142, no. 2, February 2001 (2001-02-01), pages 521 - 527, XP002220886, ISSN: 0013-7227 *
DRUCKER ET AL.: "Glucagon-like peptide I stimulates insulin gene expression and increases cyclic AMP levels in a rat islet cell line", PROC. NATL. ACAD. SCI., vol. 84, 1987, pages 3434 - 3438
E.W. MARTIN: "Remington's Pharmaceutical Sciences", MACK PUB. CO.
ELAHI ET AL.: "The insulinotropic actions of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (7-36) in normal and diabetic subjects", REGUL. PEP., vol. 51, 1994, pages 63 - 74
FEHMANN ET AL.: "Cell and Molecular Biology of the Incretin Hormones Glucagon-Like Peptide-I and Glucose-Dependent Insulin Releasing Polypeptide", ENDOCRINE REV., vol. 16, 1995, pages 390 - 410
FEHMANN ET AL.: "Insulinotropic hormone glucagon-like peptide-1 (7-37) stimulation ofproinsulin gene expression and proinsulin biosynthesis in insulinoma BTC-1 cells", ENDOCRINOLOGY, vol. 130, 1992, pages 159 - 166
GEULA; MESULAM: "Cortical cholinergic fibers in aging and Alzheimer's disease: a morphometric study", NEUROSCIENCE, vol. 33, 1989, pages 469 - 81
GHAZZI ET AL.: "Cardiac and glycemic benefits oftroglitazone treatment in NIDDM", DIABETES, vol. 46, pages 433 - 439
GOKE ET AL.: "Cardiac and Glycemic Benefits of Troglitazone Treatment in NIDDM", DIABETES, vol. 46, 1993, pages 433 - 439
GOKE ET AL.: "Distribution ofGLP-1 binding sites in the rat brain: evidence that exendin-4 is a ligand of brain GLP-1 binding sites", EUR. J. NEUROSCI, vol. 7, 1995, pages 2294 - 2300
GOKE ET AL.: "Exendin-4 is a high potency agonist and truncated exendin-4 (9-39)-amide in an antagonist at the GLP-1 (7-36)-amide receptor of insulin-secreting-cells", J. BIOL. CHEM., vol. 268, 1993, pages 19650 - 19655
GREIG N ET AL.: "Once daily injection of exendin-4 to diabetic mice achieves long-term beneficial effects on blood glucose concentrations", DIABETOLOGIA, vol. 42, 1999, pages 45 - 50
GUTNIAK ET AL.: "Antidiabetogenic effect of glucagon-like peptide-1 (7-36) amide in normal subjects and patients with diabetes mellitus", N. ENGL. J. MED., vol. 326, 1992, pages 1316 - 1322
IWAI T ET AL: "Effects of glucagon-like peptide-1 on LTP in beta-amyloid protein (1-42)-treated hippocampal slices", SOCIETY FOR NEUROSCIENCE ABSTRACTS, vol. 26, no. 1-2, 2000, & 30TH ANNUAL MEETING OF THE SOCIETY OF NEUROSCIENCE; NEW ORLEANS, LA, USA; NOVEMBER 04-09, 2000, pages - 420.7, XP001206061, ISSN: 0190-5295 *
JIN ET AL.: "Distribution of glucagonlike peptide I (GLP-I), glucagon, and glicentin in the rat brain: an immunocytochemical study", J. COMP. NEUROL., vol. 271, 1988, pages 519 - 32
LAHIRI D K ET AL: "Exendin-4 (Ex-4) revives PC12 cells from nerve growth factor (NGF)-mediated cell death and apoptosis", SOCIETY FOR NEUROSCIENCE ABSTRACTS, vol. 27, no. 2, 2001, & 31ST ANNUAL MEETING OF THE SOCIETY FOR NEUROSCIENCE; SAN DIEGO, CALIFORNIA, USA; NOVEMBER 10-15, 2001, pages 2620, XP001206062, ISSN: 0190-5295 *
LAHIRI DK; FARLOW MR; HINTZ N; UTSUKI T; GREIG NH: "Cholinesterase inhibitors, beta- amyloid precursor protein and amyloid beta-peptides in Alzheimer's disease", ACTA NEUROL SCAND, vol. 176, 2000, pages 60 - 67
LI YAZHOU ET AL: "GLP-1 receptor stimulation preserves primary cortical and dopaminergic neurons in cellular and rodent models of stroke and Parkinsonism.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 27 JAN 2009 LNKD- PUBMED:19164583, vol. 106, no. 4, 27 January 2009 (2009-01-27), pages 1285 - 1290, XP002611030, ISSN: 1091-6490 *
MARK RJ; PANG Z; GEDDES JW; UCHIDA K; MATTSON MP: "Amyloid beta-peptide impairs glucose transport in hippocampal and cortical neurons: involvement of membrane lipid peroxidation", J NEUROSCI, vol. 17, 1997, pages 1046 - 1054
MATTSON MP; LOVELL MA; FURUKAWA K ET AL.: "Neurotrophic factors attenuate glutamate-induced accumulation of peroxides, elevation of intracellular Ca2+ concentration, and neurotoxicity and increase antioxidant enzyme activities in hippocampal neurons", J NEUROCHEM, vol. 65, no. 4, 1995, pages 1740 - 1751
MERRIFIELD ET AL., J. AM. CHEM. SOC., vol. 85, 1963, pages 2149 - 2156
MOCERI ET AL.: "Early-life risk factors and the development of Alzheimer's disease", NEUROLOGY, vol. 54, 2000, pages 415 - 420
MONTROSE-RAFIZADEH C; WANG Y; JANCZEWSKI AM ET AL.: "Overexpression of glucagon- like peptide- receptor in an insulin-secreting cell line enhances glucose responsiveness", MOL CELL ENDOCRINOL, vol. 130, no. 1-2, 1997, pages 109 - 117
MONTROSE-RAFIZADEH ET AL.: "High potency antagonists of the pancreatic glucagon-like peptide- receptor", J. BIOL. CHEM., vol. 272, 1997, pages 21201 - 21206
MONTROSE-RAFIZADEH ET AL.: "Incretin hormones regulate glucose dependent insulin secretion in RIN 1046-38 cells: mechanisms of action", ENDOCRINOLOGY, vol. 135, 1994, pages 589 - 594
NATHAN ET AL.: "Insulinotropic action of glucagonlike peptide-I-(7-37) in diabetic and nondiabetic subjects", DIABETES CARE, vol. 15, 1992, pages 270 - 276
NAUCK ET AL., DIABETOLOGIA, 1993
NAUCK ET AL., J. CLIN. INVEST., 1993
NAUCK ET AL.: "Normalization of fasting hyperglycemia by exogenous glucagon-like peptide-1 (7-36) amide in type II (non-insulin dependent) diabetic patients", DIABETOLOGIA, vol. 36, 1993, pages 741 - 744
NAUCK ET AL.: "Preserved incretin activity of Glucagon-like peptide 1 (7-36) amide but not of synthetic human gastric inhibitory polypeptide in patients with Type-2 diabetes mellitus", J. CLIN. INVEST., vol. 91, 1993, pages 301 - 307
NAYA ET AL.: "Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/neuroD-deficient mice", GENES DEV., vol. 11, 1997, pages 2323 - 2334
OKA J-I ET AL: "ENDOGENOUS GLP-1 IS INVOLVED IN BETA-AMYLOID PROTEIN-INDUCED MEMORY IMPAIRMENT AND HIPPOCAMPAL NEURONAL DEATH IN RATS", BRAIN RESEARCH, ELSEVIER, AMSTERDAM, NL, vol. 878, no. 1/02, 29 September 2000 (2000-09-29), pages 194 - 198, XP001205985, ISSN: 0006-8993, DOI: 10.1016/S0006-8993(00)02741-4 *
ORSKOV: "Glucagon-like peptide-1, a new hormone of the entero-insular axis", DIABETOLOGIA, vol. 35, 1992, pages 701 - 711
OTT ET AL.: "Diabetes mellitus and the risk of dementia: The Rotterdam Study", NEUROLOGY, vol. 53, 1999, pages 1937 - 42
PAXINOS; WATSON: "NSW Australia", 1998, ACADEMIC PRESS, article "The rat brain in stereotaxic coordinates"
PERRY ET AL.: "Behavioural, histological and immunocytochemical consequences following 192 IgG-saporin immunolesions of the basal forebrain cholinergic system", BRAIN RES. BULL., vol. 54, 2001, pages 29 - 48
PERRY T A ET AL: "ENHANCING CENTRAL NERVOUS SYSTEM ENDOGENOUS GLP-1 RECEPTOR PATHWAYS FOR INTERVENTION IN ALZHEIMER'S DISEASE", CURRENT ALZHEIMER RESEARCH, BENTHAM SCIENCE PUBLISHERS, NL, vol. 2, no. 3, 1 July 2005 (2005-07-01), pages 377 - 385, XP001246463, ISSN: 1567-2050, DOI: 10.2174/1567205054367892 *
PERRY T ET AL: "NOVEL NEUROPROTECTIVE ROLE OF GLUCAGON-LIKE PEPTIDE-1 (GLP-1): IMPLICATIONS IN NEURODEGENERATIVE DISORDERS", ABSTRACTS OF THE ANNUAL MEETING OF THE SOCIETY FOR NEUROSCIENCE, SOCIETY FOR NEUROSCIENCE, WASHINGTON, DC, US, vol. 27, no. 1, 10 November 2001 (2001-11-10), pages 298, XP008072635, ISSN: 0190-5295 *
PERRY TRACYANN ET AL: "A novel neurotrophic property of glucagon-like peptide 1: A promoter of nerve growth factor-mediated differentiation in PC12 cells", JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, vol. 300, no. 3, March 2002 (2002-03-01), pages 958 - 966, XP002323789, ISSN: 0022-3565 *
RITZEL ET AL.: "Pharmacokinetic, insulinotropic, and glucagonostatic properties of GLP-1 [7-36 amide] after subcutaneous injection in healthy volunteers. Dose-response-relationships", DIABETOLOGIA, vol. 38, 1995, pages 720 - 725
SAMBROOK ET AL.: "Molecular Cloning - A Laboratory Manual", vol. 1-3, 1989, COLD SPRING HARBOR PRESS
SAMBROOK ET AL.: "Molecular Cloning, a Laboratory Manual", vol. 1-3, 1989, COLD SPRING HARBOR LABORATORY PRESS
SATOH ET AL.: "Characterization of human and rat glucagon-like peptide- receptors in the neurointermediate lobe: lack of coupling to either stimulation or inhibition of adenylyl cyclase", ENDOCRINOLOGY, vol. 141, 2000, pages 1301 - 9
SHUGHRUE ET AL.: "Glucagon-like peptide- receptor (GLP1-R) mRNA in the rat hypothalamus", ENDOCRIN, vol. 137, no. 11, 1996, pages 5159 - 62
SUZUKI N; CHEUNG TT; CAI XD; ODAKA A; OTVOS L, JR; ECKMAN C; GOLDE TE; YOUNKIN SG: "An increased percentage of long amyloid beta protein secreted by familial amyloid beta protein precursor (beta APP717) mutants", SCIENCE, vol. 264, 1994, pages 1336 - 1340
THORENS ET AL.: "Cloning and functional expression of the human islet GLP-1 receptor. Demonstration that exendin-4 is an agonist and exendin(9-39) an antagonist of the receptor", DIABETES, vol. 42, 1993, pages 1678 - 1682
THORENS ET AL.: "Glucagon-like peptide- and the control of insulin secretion in the normal state and in NIDDM", DIABETES, vol. 42, 1993, pages 1219 - 1225
WANG ET AL., ENDOCRINOLOGY, 1995
WANG ET AL.: "GIP regulates glucose transporters, hexokinases, and glucose-induced insulin secretion in RIN 1046-38 cells", MOLL. CELL. ENDO., vol. 116, 1996, pages 81 - 87
WANG ET AL.: "Glucagon-like peptide- affects gene transcription and messenger ribonucleic acid stability of components of the insulin secretory system in RIN 1046-38 cells", ENDOCRINOLOGY, vol. 136, 1995, pages 4910 - 4917
WEI ET AL.: "Tissue-specific expression of the human receptor for glucagon-like peptide-1: brain, heart and pancreatic forms have the same deduced amino acid sequences", FEBS LETT, vol. 358, no. 3, 30 January 1995 (1995-01-30), pages 219 - 224
WILMS ET AL.: "Gastric emptying, glucose responses, and insulin secretion after a liquid test meal: effects of exogenous glucagon-like peptide- (7-36) amide in Type II (non-insulin-dependent) diabetic patients", J. CLIN. EDOCRINOL. METAB., vol. 81, 1996, pages 327 - 332

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